AU2654201A - Methods for synthesis of alpha-D-GAL (1-3) GAL-containing oligosaccharides - Google Patents

Description

WO 01/51499 PCT/AU01/00028 METHODS FOR SYNTHESIS OF U-D-GAL(1-+3) GAL-CONTAINING OLIGOSACCHARIDES FIELD OF THE INVENTION 5 This invention relates to methods for synthesis of biologically active di- and tri-saccharides comprising oa-D Gal(1-+3)-D-Gal. In particular the invention provides novel reagents, intermediates and processes for the solution or solid phase synthesis of u-D-galactopyranosyl 10 (1-3)-D-galactose, and derivatives thereof. BACKGROUND OF THE INVENTION The advent of methods for successful organ transplantation has led to an increasing shortage of donor 15 organs suitable for clinical application. Immuno-concordant species such as non-human primates are potentially a source of allografts which would provide the lowest immunological barrier, but limited availability and ethical concerns, as well as the risk presented by primate retroviruses, mean 20 that this source does not provide a long term solution. Xenografts from discordant but more readily available species, such as pigs, are usually rejected almost immediately. This phenomenon is known as hyperacute rejection (HAR). Thus the suppression of xenoreactive 25 natural antibodies is a key procedure in the implementation of successful xenotransplantation (Tong, Z. et al, 1998). It has been reported that ligands comprising the non reducing terminal oligosaccharides Gala(l-+3)Gal and Gala(l-+3)Gal(1-+4)GlcNAc showed the highest affinity with 30 human anti-porcine antibodies (Good, H. et al. 1992). Of the various means proposed for overcoming HAR, the simplest in concept are the competitive blocking of Galax(l-+3)Gal antibodies in vivo, or the extracorporeal removal of these antibodies from the circulation (Simon, P.M., 1996). Both 35 methods require the ready availability of the disaccharide or trisaccharide.

WO 01/51499 PCT/AU01/00028 -2 In addition to this problem, intestinal infection by Clostridium difficile is one of the most common causes of diarrhoea in hospital patients, especially in the elderly (Boriello, S.P., 1990). C. difficile has been found to be 5 an aetiological agent of antibiotic-associated diarrhoea and pseudomembranous colitis (Smith, J.A. et al., 1997). C. difficile produces two toxins, toxin A and toxin B. Of these, toxin A was shown in animal studies to be an enterotoxin that elicits increased intestinal permeability, 10 fluid secretion and inflammation, and causes severe disruption of the intestinal epithelium (Burakoff, R. et al, 1995; Castex, F. et al, 1994; Eglow, R. et al., 1992; Torres, J. et al, 1990). In model animal systems, the carbohydrate moiety to which toxin A binds has been shown 15 to terminate in the trisaccharide sequence GalOa(1-+3)GalP(1-+4)GlcNAc (Krivan, H.C. et al, 1986). Although the chemistry and biochemistry of oligosaccharide compounds has been extensively studied, there are still difficulties associated with their 20 synthesis and purification. Consequently there is a need in the art for improved methods of synthesis and purification of these compounds. Apart from the design of effective building blocks, one of the most difficult steps in the synthesis of 25 Gala(1-+3)Gal, GalX(1-+3 )Gal@(1-+4)GlcNAc and related compounds is the formation of the a(1-+3) linkage. Although a number of synthetic routes have been described, all of these methods are complex, time-consuming, and costly, and are unsuited to large-scale synthesis. 30 Chacon-Fuertes provided a procedure for the synthesis of 3-0-a-D-galactopyranosyl-D-galactose [ii WO 01/51499 PCT/AUO1/00028 -3 OH OH 0 OH OH HO OH O HOO OH O OH OH (i) which required a mercuric cyanide-catalysed glycosylation 5 for formation of the a(l-+3) glycosidic linkage (Chacon Fuertes M.E. and Martin-Lomas, M., 1975). The synthesis was protracted, required chromatography, and used dangerous reagents. Lemieux described the chemical synthesis of 3-0--a-D 10 galactopyranosyl-D-galactose using a per-O-benzylated cC-D galactopyranosyl bromide sugar donor and a 2,2,2-trichloroethyl 2,4, 6-tri-O-acetyl-3-D-galact opyranoside acceptor (Lemieux, R.U. and Driguez, H., 1975). Lemieux employed tetraethyl ammonium bromide as a promoter 15 in a reaction that after chromatography gave 35% yield of product. H NMR spectroscopy indicated that the glycosylation product still contained substantial impurities. After deprotection with zinc/acetic acid and preparative thin layer chromatography, de-O-acetylation, 20 hydrogenolysis and paper chromatography, an authentic sample of 3-O-a-D-galactopyranosyl-D-galactose was finally achieved. An alternative approach used an allyl 2-0-benzoyl 4, 6-0-benzylidene--D-galactopyranoside acceptor and an 25 acetimidate sugar donor (Sinay, P. and Jacquinet, J.C., 1979) . The formation of the x(1-+3) linkage was effected with toluene sulphonic acid in nitromethane in good yield, but chromatography was required for purification. Although generally maintaining yields of greater than 90%.for the 30 remainder of the synthesis to the target 3-0-a-D galactopyranosyl-D-galactose, chromatography was required WO 01/51499 PCT/AUO1/00028 -4 at most steps. Similarly a benzylated Gal(cal-3)Gal disaccharide was synthesised using an c-D-galactopyranosyl bromide donor, but employing stannylene chemistry to selectively activate the 3-0-position of the acceptor 5 galactoside, (Auge, C. and Veyrieres, A., J.C.S., 1979). The benzylated Gala (1-+3) Gal disaccharide subsequently underwent hydrogenolysis to afford 3-0-L-D galactopyranosyl-D-galactose. The reported yields were very low, and most steps required chromatography. 10 Another synthesis of the 3-0-a-D-galactosyl-D galactose disaccharide employed a benzyl 2,4,6-tri-0 benzyl-P-D-galactopyranoside acceptor and a fully benzylated imidate galactosyl donor (Milat, M-L. et al, 1982). The free disaccharide was eventually obtained after 15 a final hydrogenolysis, and although reasonable yields were achieved, chromatography was unavoidable at many stages of the synthesis. Takeo employed a galactosyl bromide donor and tetraethylammonium bromide as a promoter, and synthesised the disaccharide of interest in a protected 20 form in 40% yield after chromatography. Hydrogenolysis then yielded 3-0-ca-D-galactopyranosyl-D-galactose (Takeo, K. and Maeda, H., 1988). A chemo-enzymatic synthesis utilised X-D-galactosidase from coffee beans to form the disaccharide, in unreported yield. p-Nitrophenyl-a-D 25 galactopyranoside was used as both the acceptor and donor. The resultant disaccharide derivative was then modified and chromatographed to afford 3-0-a-D-galactopyranosyl-D galactose (Matsuo, I. et al, 1997). It is desirable to avoid the use of toxic reagents, 30 and in order to reduce costs it is also highly desirable to minimise the number of purification steps. If possible, it is particularly desirable to minimize the number of chromatographic purification steps, or even to avoid entirely the need for chromatographic purification, because 35 this technique is time-consuming and costly. Synthesis of the trisaccharide a-D-galactopyranosyl (1-+3) -0-D-galactopyranosyl- (1-+4) -N-acetyl-D-glucosamine WO 01/51499 PCT/AUO1/00028 -5 (ii) has understandably been even more difficult than that of a-D-galactopyranosyl- (1-+3) -D-galactose. OH OH O OH OH HO O AcHN OH OH HO OH 0 OO OHH (ii) 5 There have been no methods reported in the literature for the synthesis of (ii) using chemical means, although closely analogous compounds have been developed for in vitro and in vivo applications (Garegg, P.J. and Oscarson, 10 S., 1985; Schaubach, R. et al, 1991). There have been some reports of enzymatic synthesis of oligosaccharide (ii) and derivatives thereof. Nilsson synthesised the 2-N trichloroethoxycarbonyl protected ethyl thioglycoside of (ii) by enzymatic methods, using an a-D-galactosidase to 15 effect the formation of the x(1-+3) glycosidic linkage followed by -D-galactosidase treatment (Nilsson, K.G.I., 1997). Similarly galactosidases have been used for the synthesis of target compound (ii), employing similar methodologies (Matsuo, I. et al, 1997) . Another ethyl 20 thioglycoside derivative of (ii) was synthesised using a and P galactosidases (Vic, G. et al, 1997) . Analogues of (ii) similar to those described above with lipophilic tails attached via the glycosidic linkage were synthesised using X(1->3) galactosyltransferases (Sujino, K. et al., 1998). 25 All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the 30 applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly WO 01/51499 PCT/AU01/00028 -6 understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in 5 Australia or in any other country. We have now found that novel thioacyl-substituted glycosides of 3-0-aX-D-galactopyranosyl-D-galactose can be used for glycoconjugate synthesis by chemical methods. These derivatives can be linked to a suitable solid 10 support, such as polyethylene .glycol. These compounds can be used for removal of anti-Gal antibodies from a transplant recipient's blood prior to xenotransplantation, or as anti-bacterial agents to combat bacteria such as C. difficile. 15 SUMMARY OF THE INVENTION In a first aspect the invention provides a protected glucosamine compound of general formula I: R2 0 OBn HO V -,NHRl OBn 20 in which R 1 is H or acetyl and R 2 is benzyl or 4 chlorobenzoyl, 25 with the proviso that when R2 is benzyl, R1 is not acetyl. In a second aspect, the invention provides a protected monosaccharide building block of general formula II: 0 SMe 0 "OR' 2

R

3

.

OR

WO 01/51499 PCT/AU01/00028 -7 in which R 3 is H, methoxy or methyl, and in which (a) when R 3 is methoxy or methyl, R' is H, benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 3,4- I1 5 methylenedioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4 acetamidobenzyl, or 4-azidobenzyl; and

R

2 is H, Fmoc, benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 3,4-methylenedioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 10 4-acetamidobenzyl, or 4-azidobenzyl; (b) when R 3 is H, R1 is benzoyl, pivaloyl, 4 chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, benzyl, 3,4-methylene-dioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4-acetamidobenzyl, or 4-azidobenzyl, and 15 R 2 is Fmoc, benzoyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4 acetamidobenzyl, or 4-azidobenzyl, with the provisos that 20 (i) when R is acetyl, R2 is not chloroacetyl or acetyl, and vice versa; (ii) when R 2 is levulinoyl, R' is not benzoyl, and vice versa; and (iii) when R' is benzoyl, R 2 is not benzoyl, and vice 25 versa. When R 2 is Fmoc, R' is benzoyl, pivaloyl, 4 chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4 methylbenzoyl, benzyl, 3,4-methylene-dioxybenzyl, 4 methoxybenzyl, 4-chlorobenzyl, 4-acetamidobenzyl, or 4 30 azidobenzyl. Preferably the compound is of general formula III: O 0 SMe O- " OR 111

OR

2 WO 01/51499 PCT/AU01/00028 -8 in which R' is pivaloyl, benzoyl, 4-chlorobenzoyl, 4 methoxybenzyl, or 3,4-methylenedioxybenzyl, and R2 is H, Fmoc, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methoxybenzyl, or 3, 4-methylenedioxybenzyl, 5 with the proviso that if R' is benzoyl, R 2 is not levulinoyl. In preferred embodiments, the compound is (a) a galactopyranoside of general formula III, in which R 1 is 4-chlorobenzoyl, pivaloyl or acetyl, and R 2 is 10 Fmoc or H; (b) a compound of general formula III in which R' is 4-chlorobenzoyl and R 2 is chloroacetyl; or (c) a compound of general formula III in which both R 1 and R2 are 3,4-methylenedioxybenzyl. 15 In a third aspect, the invention provides a galactopyranoside compound of general formula IV: R1O 0 SMe

R

1 0 "OR IV OR in which each R' is independently 4-chlorobenzyl, 4 20 azidobenzyl, 4-N-acetamidobenzyl, 4-methylbenzyl, 3,4 methylenedimethoxybenzyl, or 2-nitrobenzyl. Preferably each R' is 4-chlorobenzyl. In a fourth aspect the invention provides a polyethyleneglycol (PEG)-linked monosaccharide of general 25 formula V: 0 0 OnR1\ 0 ~ 0 SORO R in which n is an integer from 1-5; WO 01/51499 PCT/AU01/00028 9 R' is a linking group or a group suitable for the formation of a covalent linkage, and includes but is not limited to groups such as halogen, azido, carboxylic acid, thiol, hydroxyl, thioester, xanthate, amido, or 5 dithiocarbamate; R 2 is acetyl, 4-chlorobenzoyl, levulinoyl, pivaloyl, chloroacetate, benzoyl, or 4-methybenzoyl;

R

3 is H, Fmoc, benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, 10 or 4-azidobenzyl; and

R

4 is methoxy, H, or methyl. Preferably n is 2, R 1 is thiobenzoate or thiobiphenylcarbonyl, R 2 is 4-chlorobenzoyl, R 3 is H, and R 4 is H. 15 In a fifth aspect the invention provides a compound of general formula VI: R40 0

OR

1 O eR3 0 "R 0 3 W. R

OR

6 VI in which R 7 is H, methoxy or methyl; R1 is aryl, substituted aryl, benzyl, substituted 20 benzyl, alkyl, substituted alkyl, PEG, or substituted PEG; R2 is acetamido or amino;

R

3 and R 4 are independently benzyl, substituted benzyl, silylether or acyl;

R

5 is 4-chlorobenzoyl, benzoyl, pivaloyl, acetyl, 25 levulinoyl or 4-methylbenzoyl; and R6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3, 4-methylenedioxybenzyl, 4-methoxybenzyl, 4 acetamidobenzyl, or 4-azidobenzyl.

WO 01/51499 PCT/AUO1/00028 - 10 When the anomeric configuration of the glucosamine moiety of general formula VI is a and R 3 is benzyl and R 4 is benzoyl and R7 is H, then R2 may be acetamido, amino, N phthalimido, R 5 may be 4-chlorobenzoyl, benzoyl, pivaloyl, 5 acetyl, levulinoyl or 4-methylbenzoyl, and R 6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl. 10 When the anomeric configuration of the glucosamine moiety of general formula VI is P and R is benzyl and R 7 is H, then R 2 is acetamido, amino, or N-phthalimido; R 3 and

R

4 are independently benzyl, substituted benzyl, silylether or acyl; R 5 is 4-chlorobenzoyl, benzoyl, pivaloyl, acetyl, 15 levulinoyl or 4-methylbenzoyl, and R 6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3,4-methylenedioxybenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl. When the anomeric configuration of the glucosamine 20 moiety of general formula VI is a and R', R 3 , and R 4 are benzyl or substituted benzyl and R 7 is H, then R 2 is acetamido, amino, or N-phthalimido, R 5 is pivaloyl, 4 chlorobenzoyl, benzoyl, or levulinoyl, and R6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, 25 H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl, with the proviso that when R 3 and R 4 are benzyl, R 5 is not acetyl or benzoyl. In preferred embodiments: 30 (a) the anomeric configuration of the glucosamine moiety of general formula VI is P, R3 is benzyl, R2 is amino or acetamido, R 3 and R 4 are benzyl, R 5 is 4-chlorobenzoyl, pivaloyl or acetyl, R 6 is Fmoc or H, and R 7 is H; (b) the anomeric configuration of the glucosamine moiety 35 of general formula VI is (X, R is benzyl, R 2 is acetamido,

R

3 is benzyl, R 4 is benzoyl or benzyl, R 5 is 4 chlorobenzoyl, R 6 is H or 4-chloroacetyl and R 7 is H; WO 01/51499 PCT/AU01/00028 - 11 (c) the compound is a trisaccharide of General Formula VII: Ph O OR' CIBnO 0 0 OBn CIBnO -- "O 0"O NHR OR OBn CIBnO OBnCI VII 5 in which R is H or acetyl; R is hydrogen, benzyl, benzoyl or p-chlorobenzoyl; and R 2 is hydrogen, 4-chloro-benzoyl, acetyl, benzoyl or pivaloyl; (d) the compound is a trisaccharide of general formula 10 VII, in which the anomeric configuration of the reducing end is (X, R is acetyl, R' is benzoyl, 4-chlorobenzoyl or H, and R2 is 4-chlorobenzoyl or H; or (e) the compound is a trisaccharide of general formula VII, in which the anomeric configuration of the reducing 15 sugar is P, R is acetyl or H, R 1 is benzyl, and R 2 is H, 4 chlorobenzoyl, pivaloyl or acetyl. In a sixth aspect the invention provides a compound of general formula VIII:

R

4 0 0 R 6 0 ...0 0 VIII

OR

5 OR 5 OR 2 X-R' 20 in which R.

5 , R 6 and R.

7 are independently H, 4 chlorobenzyl, 4-methoxybenzyl, 4-methylbenzyl, 4 acetamidobenzyl, azidobenzyl or 3, 4-methylenedioxybenzyl; X is 0, S, or N; WO 01/51499 PCT/AUO1/00028 - 12 R 1 is alkyl, substituted alkyl, aryl, substituted aryl, PEG or substituted PEG;

R

2 is levulinoyl, 4-chlorobenzoyl, benzoyl, 4 methylbenzoyl, acetyl or pivaloyl; and 5 R 3 and R 4 may combine to form a benzylidene ring, which may optionally be substituted at the 4 position by methyl or methoxy; alternatively R 3 and R 4 may independently be H, benzyl or substituted benzyl. When R 5 is 4-chlorobenzyl, 4-methoxybenzyl, 4 10 methylbenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4 methylenedioxybenzyl, and R and R7 combine to form a benzylidene or substituted benzylidene ring, then X is 0, S, or N, R is alkyl, substituted alkyl, aryl, substituted aryl, PEG, substituted PEG, acyl or substituted acyl, and 15 R 2 is levulinoyl, 4-chlorobenzoyl, benzoyl, 4 methylbenzoyl, acetyl or pivaloyl. When X is oxygen and R' is 3,4-methylenedioxybenzyl, then R2 is H, 4-chlorobenzoyl, pivaloyl, acetyl, levulinoyl, benzoyl or chloroacetyl, R 3 and R 4 may combine 20 to become a benzylidene ring or may independently be H, benzyl or substituted benzyl, and R 5 , R 6 and R7 may be H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl. When X is oxygen and R 1 is 2-[2-(2-thiobenzoyl) 25 ethoxy)ethyl or 2-[2-(2-thiobiphenylcabonyl)ethoxy], then R2 is H, 4-chlorobenzoyl, pivaloyl, acetyl, levulinoyl, benzoyl or chloroacetyl, R 3 and R 4 may combine to form a benzylidene ring or may independently be H, benzyl, 4 chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, 30 azidobenzyl or 3,4-methylenedioxybenzyl, R 5 is H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl, and R 6 and R 7 may combine to become a benzylidene ring or may independently be H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4 35 acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl. When X is sulphur, R' is alkyl, substituted alkyl, aryl or substituted aryl, R 3 and R 4 combine to form a WO 01/51499 PCT/AU01/00028 - 13 benzylidene ring and R 5 , R 6 and R 7 are benzyl, then R 2 is levulinoyl, 4-chlorobenzoyl, benzoyl, acetyl or pivaloyl, with the proviso that when R' is phenyl, R 2 is not levulinoyl. 5 Preferably either (a) X is oxygen, R 1 is 2-[2- (2-thiobenzoyl)ethoxy)ethyl or 2-[2-(2-thiobiphenylcabonyl)ethoxyl, R2 is H or 4 chlorobenzoyl, R 3 and R 4 are H or combine to form a benzylidene ring, R 5 is H or 3,4-methylenedioxybenzyl, and 10 R 6 and R 7 are both H or combine to form a benzylidene ring; (b) X is S, R 1 is methyl, R 2 is 4-chlorobenzoyl, R 3 and R 4 combine to form a benzylidene ring, and R, R 6 and R 7 are each 4-chlorobenzyl; or (c) X is oxygen, R' is 3,4-methylenedioxybenzyl, R 2 is 4 15 chlorobenzoyl or H, R 3 and R 4 combine to form a benzylidene ring or are both H, and R 5 , R 6 and R 7 are independently 4 chlorobenzyl or H. In a seventh aspect the invention provides a compound of general formula IX: 0 0

R

4 0 0 0

R

3 0 'OR 1 IX OR2 20 in which R 1 is 4-chlorobenzoyl, pivaloyl, acetyl, levulinoyl, benzoyl or chloroacetyl; R2 is H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4 acetamidobenzyl, azidobenzyl, 3,4-methylenedioxybenzyl, 25 Fmoc, levulinoyl, acetyl or chloroacetyl; and

R

3 and R 4 may combine to form a benzylidene ring, or may independently be H, benzyl, 4-chlorobenzyl, 4 methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4 methylenedioxybenzyl.

WO 01/51499 PCT/AUO1/00028 - 14 Preferably R 1 is 4-chlorobenzoyl, R 2 is H, and R 3 and

R

4 combine to form a benzylidene ring. In an eighth aspect the invention provides a polyethyleneglycol (PEG) -linked disaccharide of General 5 Formula X or a trisaccharide of General Formula XI: OH OH OH OH HO OH O 0 R 0 0 0 S' OH HO O AcHN )10 0 xO nO OH OH O OH OH HO o

O

0 Onj OH n XI 10 in which R is hydrogen or acyl, and n is an integer of from 1 to 3. Preferably the compound of General Formula XI is 2 [2- (2-thiobiphenylcarbonyl) ethoxy] -ethyl 3-0- (cX-D 15 galactopyranosyl)-a-galactopyranoside. In a ninth aspect, the invention provides GalcL(l->3)Gal$ (l->4 )GlcNAc coupled to a solid support to give a compound of general formula XII: WO 01/51499 PCT/AUO1/00028 - 15 0 HO O0 NH NH- (C2sSC2 H(H)OX O'" "NHAc OH OH HO OH oi,...~ OH OH OH OH 0 OH XII 5 in which X is a solid support such as Sepharose or silica gel, and n is an integer of from 3 to 6. The compounds of the first seven aspects of the invention are useful as intermediates in the synthesis of 10 di- and trisaccharides. Accordingly, in a tenth aspect, the invention provides a method of synthesis of a desired compound of General Formula X to General Formula XII, or of a-D-galactopyranosyl- (1->3) -0-D-galactopyranosyl- (1-+4) -N acetyl-D-glucosamine (Gal (1->3 )Galp (1-4)GlcNAc), a-D 15 galactopyranosyl- (1-+3) -p-D-galactopyranose (Gal (l-+3 )Gal), or -D-galactopyranosyl- (1-+4) -N-acetyl D-glucosamine (Gal$(1--4)GlcNAc), comprising the step of using a compound of General Formula I to IX as an intermediate. 20 Preferably when the desired compound is of general Formula X or XI the intermediate compound is of General Formula V. It will be clearly understood that although a compound of General Formula VI may be synthesised using a compound of General Formula I as an intermediate, 25 alternative syntheses are available. For the purposes of this specification, the term "alkyl" is intended to include saturated, unsaturated and cyclic hydrocarbon groups, and combinations of such groups.

WO 01/51499 PCT/AUO1/00028 - 16 Suitable substituents on hydrocarbon chains or aryl rings include Br, Cl, F, I, CF 3 , NH 2 , substituted amino groups such as NHacyl, hydroxy, carboxy, Ci-salkylamino and Ci 6 alkoxy groups such as methoxy, and are preferably F, Cl, 5 hydroxy, Ci- 6 alkoxy, amino, C 1

-

6 alkylamino or C 1

-

6 carboxy. In a eleventh aspect, the invention provides a method of preventing or reducing a hyperacute rejection response associated with xenotransplantation, comprising the step of administering an effective dose of thioalkyl Gala-(1-+3)Gal 10 or thioalkyl Gala(1-+3)Gal3(1-+--4)GlcNAc to a subject in need of such treatment. The compound may be administered before, during or after xenotransplantation. In a twelfth aspect, the invention provides a method 15 of preventing or reducing hyperacute rejection associated with xenotransplantation, comprising the steps of a) removing plasma from a patient who is to undergo xenotransplantation; b) exposing the plasma to thioalkyl Galcx(l-+3)Gal or 20 thioalkyl Gala(l->3)Gal$(l--->4)GlcNAc linked to a solid support, and c) reinfusing the thus-treated plasma into the patient. In a thirteenth aspect, the invention provides a 25 method of depleting anti-Galx(l-+3)Gal antibodies from a plasma or serum sample, comprising the step of exposing the plasma or serum to thioalkyl GalcL(1-+3)Gal or thioalkyl Gala(1-->3)Galp (1-+4)GlcNAc linked to a solid support. In a fourteenth aspect, the invention provides a 30 method of treatment of C. difficile infection, comprising the step of administering an effective amount of a-D galactopyranosyl- (1-+3) -p-D-galacto-pyranosyl- (1-+4) -N acetyl-D-glucosamine (Galac(l-+3)Gal$(1-+4)GlcNAc) or of thioalkyl Gala(1-+-3)Gal$ (1-+4)GlcNAc, preferably linked to 35 a solid support, to a subject in need of such treatment. Preferably the solid support is a multidentate ligand or a dendrimer compound. Suitable dendrimers are disclosed WO 01/51499 PCT/AUO1/00028 - 17 for example in International patent application No. PCT/AU95/00350 (WO95/34595) by Biomolecular Research Institute Ltd. In the eleventh to the fourteenth aspects of the 5 invention, the subject may be a human, or may be a domestic, companion or -zoo animal. While it is particularly contemplated that the compounds of the invention are suitable for use in medical treatment of humans, they are also applicable to veterinary treatment, including 10 treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as felids, canids, bovids, and ungulates. Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well-known 15 in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pennsylvania, USA. The compounds and compositions of the invention may be administered by any suitable route, and the person 20 skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and 25 general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered. The carrier or diluent, and other excipients, will depend on the route of administration, and again the 30 person skilled in the art will readily be able to determine the most suitable formulation for each particular case. For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word 35 "comprises" has a corresponding meaning.

WO 01/51499 PCT/AUO1/00028 - 18 DETAILED DESCRIPTION OF THE INVENTION The invention will now be described in detail by way of reference only to the following non-limiting examples. 5 Abbreviations used herein are as follows: AcN Acetonitrile Bn Benzyl

CH

2 Cl 2 Dichloromethane CHCl 3 Chloroform 10 pClBn pa-ra-chlorobenzyl pClBz para-chlorobenzoyl DCM Dichloromethane DMF N, N'-Dimethylformamide DMTST Dimethyl(methylthio)sulphoniumtrifluoro 15 methanesulphonate EtOAc Ethyl acetate EtOH Ethanol

H

2 0 Water HRMS High resolution mass spectrometry 20 MDBn 3,4-methylenedioxybenzyl Me Methyl MeCN Acetonitrile MeOH Methanol MgSO 4 Magnesium sulphate 25 NaHCO 3 Sodium.hydrogen carbonate NMR Nuclear magnetic resonance, PEG Polyethylene glycol Ph Phenyl SOCl 2 Thionyl chloride 30 TBDMS tertiary-butyldimethylsilyl THF Tetrahydrofuran Example 11: Preparation of 3,4-Methylenedioxybenzyl 4,6-0 Benzylidene 2-0-(4-chlorobenzoy1)-/J-D 35 Galactopyranoside Acceptor The strategy for this preparation is set out in Reaction Scheme 1.

WO 01/51499 PCT/AUO1/00028 - 19 Synthesis of a-D-Galactopyranosyl- (1->3) -D-Galactose OH OH OH OTBDMS H 3 C 0 OTBDMS HO SMe HO SMe H 3 CO OSMe OH OH OH 1 2 3 0 O HO OH H 3 C 0 OTBDMS HO OSHOSMe H3CO SMe OBzC OBzCi OBzC 6 5 4 0 CIBnO OBnCI 0 00 O\ CIBnO.... SMe CIBnO OBnC 0O 0 OBnCI 0 0 HO O"_,6 0 ____ CIBnO 0 OBzCO CIBnO O OBzCl 7 OH OH CIBnO OOBnC 0 HO IO O CIBnO O OH OH 11 10 OH OH O OH OH HO O OH OH 12 5 Reaction Scheme 1 WO 01/51499 PCT/AUO1/00028 - 20 Methyl 6-0-tert-butyldimethylsilyl-1-thio-Q-D galactopyranoside (2) A mixture of t-butyldimethylsilyl chloride (68.35 g, 453.51 mmol) and 4-dimethylaminopyridine (55.40 g, 453.51 5 mmol) in dry 1,2-dichloroethane (800 ml) was stirred at 80 0 C for 15 minutes. Methyl 1-thio-$-D-galactopyranoside (1) (100 g, 476.19 mmol) was added in 5 portions in 15 minutes to the stirred solution at 80 0 C, and the reaction mixture was stirred under reflux for 1 hour. The resulting 10 clear solution was cooled to room temperature, diluted with CHCl 3 (2 000 ml), washed four times with diluted brine solution (water-brine 2:1) (750 ml). The aqueous layers of the last two washings were collected and extracted with CHCl 3 (400 ml). The organic layers were combined, dried 15 over MgSO 4 and evaporated. The residue was kept under high vacuum for 15 min, then was dissolved in dry MeCN (200 ml). The solution was evaporated, and the residue was kept under high vacuum for 15 min. This drying process was repeated using another 200 ml of dry MeCN, to give the crude methyl 20 6-0-tert-butyldimethylsilyl-l-thio- -D-galactopyranoside (2) (117.5 g, 80%) as a syrup. Rf 0.65 (MeCN/H 2 0 10:1) MS (electrospray) C 13

H

28 0 5 SSi (324.23) m/z (%) 347[M+Na]* (100), 325[M+H] (75). 25 Methyl 6-O-tert-butyldimethylsilyl-3,4-0-isopropylidene-1 thio-0-D-galactopyranoside (3) A mixture of crude methyl 6-0-tert butyldimethylsilyl-l-thio-3-D-galactopyranoside (2) 30 (117.46 g, 362.27 mmol), 2,2-dimethoxypropane (66.82 ml, 543.41 mmol) and p-toluenesulphonic acid (200 mg) in dry MeCN (800 ml) was stirred at 40'C for 30 minutes. The reaction mixture was neutralized with triethylamine (3 ml) and evaporated to give a white crystalline residue (3) 35 (161.3 g).

WO 01/51499 PCT/AUO1/00028 - 21 Rf 0.62 (EtOAc/Hexane 2:1) MS (electrospray) C 16

H

32 0 5 SSi (364.58) m/z (%) 387[M+Na]* (45), 365 M+H]* (100). Methyl 6-O-tert-butyldimethylsilyl-2-0-(4-chlorobenzoyl) 5 3,4-0-isopropylidene-l-thio-#-D-galactopyranoside (4) A mixture of methyl 6-O-tert-butyldimethylsilyl-3,4 0-isopropylidene-l-thio- -D-galactopyranoside (3) (155.44 g, 427. 03 mmol) and 4-dimethylaminopyridine (62.60 g, 512.44 mmol) in dry 1,2-dichloroethane (750 ml) was stirred 10 at room temperature. 4-Chlorobenzoyl chloride (89.68 g, 512.44 mmol) was added to the stirred reaction mixture in 15 minutes. After the addition the resulting suspension was stirred under reflux for 30 minutes. The reaction mixture was cooled to 10'C and filtered. The crystalline solid was 15 washed on the funnel with dry 1,2-dichloroethane (300 ml) and filtered. The filtrates were combined, diluted with CHCl 3 (2000 ml) and washed twice with diluted brine solution (water-brine 2:1) (1500 ml). The organic layer was dried over MgSO 4 and evaporated. The residue was kept under 20 high vacuum for 15 minutes. The resulting syrup was dissolved in dry MeCN (200 ml) and evaporated using high vacuum at the end of the evaporation, to give the crude methyl 6-0-tert-butyldimethylsilyl-2-0-(4-chlorobenzoyl) 3,4-0-isopropylidene-l-thio- -D-galactopyranoside (4) (165 25 g) as a colourless syrup. Rf 0.68 (Hexane/EtOAc 2:1) MS (electrospray) C 23

H

35

O

6 SSi (503.14), m/z (%) 503[M+H]* (100), 525[M+Na]* (38). 30 Methyl 2-0-(4-chlorobenzoyl)-l-thio-#-D-galactopyranoside (5) A mixture of methyl 6-O-tert-butyldimethylsilyl-2-0 (4-chlorobenzoyl)-3,4-isopropylidene-$-D-galactopyranoside (4) (173 g, 344.62 mmol) and p-toluenesulphonic acid (600 35 mg) in MeOH-MeCN 3:1 (2000 ml) was stirred under reflux for 1 hour. The reaction mixture was cooled to room temperature and evaporated. The resulting white solid residue was WO 01/51499 PCT/AUO1/00028 - 22 suspended in diisopropylether (1000 ml) and filtered. The crystalline solid was washed twice with diisopropylether (300 ml), then with diethylether (500 ml) and dried to give methyl 2-0-(4-chlorobenzoyl)-1-thio- -D-galactopyranoside 5 (5) (107 g) as a white crystalline powder. Rf 0.45 (MeCN/H 2 0 10:1) MS (electrospray) C1 4

H

17 C10 6 S (348.80) m/z (%) 371[M+Na]* (35), 349[M+H ] (100). 10 Methyl 2-0- (4-chlorobenzoyl) -4, 6-0-benzylidene-l-thio-Q-D galactopyranoside (6) A mixture of methyl 2-0-(4-chlorobenzoyl)-l-thio- -D galactopyranoside (5) (94.16 g, 270.57 mmol), a,a-dimethoxytoluene (60.9 ml, 405.86 mmol) and 15 p-toluenesulphonic acid (200 mg) in dry MeCN (500 ml) was stirred at 70'C for 30 minutes. The reaction mixture was cooled to room temperature, neutralized with triethylamine (3 ml) and evaporated. The residue was taken up in CHCl 3 (1500 ml), washed with diluted brine solution (water- brine 20 2:1) (750 ml), with saturated NaHCO 3 solution (500 ml), with diluted brine again (water-brine 2:1) (750 ml), dried over MgSO 4 and evaporated. The resulting white solid was kept under high vacuum for 15 minutes. The dry residue was crystallized from MeCN (250 ml) at room temperature to give 25 68.5 g pure product. Water (80 ml) was added slowly to the mother liquor, and the solution was left at room temperature to crystallize another 20.8 g of methyl 2-0-(4 chlorobenzoyl)-4,6-0-benzylidene-l-thio- -D galactopyranoside (6) (yield: 75%). 30 Rf 0.65 (EtOAc/Hexane 2:1) MS (electrospray) C 21

H

21 C10 6 S (436.91) m/z (%) 437[M+H]* (56), 459[M+Na]* (100). 1H NMR (CDCl 3 ) 6 8.01-7.37 (9H, aromatic), 5.56 (s, 1H, 35 benzylidene), 5.44 (t, 1H, H-2), 4.5 (d, 1H, Ji- 2 =9.0, H-1), 4.38 (dd, 1H, H-6a), 4.30 (dd, 1H, H-4), 4.04 (dd, 1H, H- WO 01/51499 PCT/AUO1/00028 - 23 6b), 3.90 (m, 1H, H-3), 3.6 (s, iH, H-5), 2.25 (s, 3H, S

CH

3

)

3,4-Methylenedioxybenzyl 4,6-O-benzylidene 2-0-(4 5 chlorobenzoyl) -Q-D-galactopyranoside (7) To a mixture of methyl 4,6-0-benzylidene 2-0-(4 chlorobenzoyl)-l-thio- -D-galactopyranoside (6) (10 g, 22.9 mmol), 3,4-methylenedioxybenzyl alcohol (5.6 g, 36.8 mmol) and powdered molecular sieves (5A, 15 g) in dry 10 1,2-dichloroethane (200 mL) at 0 0 C, was added methyl trifluoromethanesulphonate (6 g, 36.6 mmol) in one portion under nitrogen atmosphere. The reaction mixture was sealed and left to warm to room temperature, and stirred for 3 h. The mixture was then neutralized with triethylamine 15 (15 mL), diluted with CHC1 3 (350 mL) and filtered through celite. The filtrate was washed with saturated NaHCO 3 solution (4 x 500 mL), and the organic layer was dried over MgSO 4 and evaporated to dryness leaving a white solid. The solid was suspended in diisopropylether (200 mL), filtered, 20 washed with diisopropylether (200 mL) and dried to give 3,4-methylenedioxybenzyl 4,6-0-benzylidene 2-0-(4 chlorobenzoyl)-3-D-galactopyranoside (7) (7.5 g, 61% yield) as a white powder. 25 Rf 0.60 (CH 2 Cl 2 /EtOH 20:1) MS (electrospray) C 28

H

25 ClO9 (540.95) m/z (%) 437[M+H] 4 (56), 558[M+H+NH 3 ]+ (100). Example 2: Preparation of Methyl 2,3,4,6-tetra-O-(4 chlorobenzyl)-l-thio-/-D-Galactopyranoside 30 Glycosyl Donor Methyl 2,3,4,6-tetra-O-(4-chlorobenzyl)-l-thio-3-D galactopyranoside (8) To a stirred suspension of sodium hydride (95%) (14.43 g, 571.42 mol) in dry DMF (300 ml) a solution of 35 methyl 1-thio-o-D-galactopyranoside (1) (20 g, 95.23 mmol) in dry DMF (200 ml) was added dropwise at 0 0 C in nitrogen atmosphere. At the end of the addition the ice-bath was WO 01/51499 PCT/AUO1/00028 - 24 removed and the reaction mixture was stirred at room temperature for 30 minutes. 4-Chlorobenzyl chloride (97.74 g, 571.42 mmol) was added dropwise to the stirred reaction mixture keeping the temperature 10-20 0 C. After the 5 addition, the reaction mixture was stirred at room temperature overnight. The resulting suspension was cooled with ice-bath and methanol (11 ml) was added slowly. When the hydrogen formation had stopped, the suspension was evaporated to dryness at 45-50'C. The remaining DMF was 10 removed by co-evaporation with xylene (100 ml). The residue was taken up in CH 2 C1 2 (500 ml), washed twice with water (500 ml), saturated NaHCO 3 solution (500 ml), dried over MgSO 4 and evaporated. The residue was crystallized from EtOH (500 ml) to give methyl 2,3,4,6-tetra-0 15 (4-chlorobenzyl)-l-thio- -D-galactopyranoside (8) (40 g, 60%) as a white crystalline solid. Rf 0.72 (Hexane/EtOAc 3:1) MS (electrospray) C 35

H

34 C1 4 0 5 S (708.53) m/z (%) 709[M+H]* (100), 731[M+Na]* (48). 20 Example 3: Preparation of 3-O-(a-D-galactopyranosyl)-D galactopyranose 3, 4-Methyl enedioxybenzyl 4, 6-0-benzylidene-2 -0- (4 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D 25 galactopyranosyl) -/-D-galac.topyranoside (9) Methyl trifluoromethanesulphonate (4 g, 24 mmol) was added under nitrogen to a mixture of 3,4 methylenedioxybenzyl 4,6-0-benzylidene 2-0-(4 chlorobenzoyl)- -D-galactopyranoside (7) (6.5 g, 12 mmol), 30 methyl 2,3,4,6-tetra-O-(4-chlorobenzyl)-thio- -D galactopyranoside (8) (12 g, 17 mmol) and powdered molecular sieves (5A, 10 g) in dry 1,2-dichloroethane (250 mL). The sealed reaction mixture was left to warm to room temperature and then stirred for 80 minutes. The 35 reaction mixture was neutralized with triethylamine (12 g) and diluted with CHCl 3 (500 mL). The suspension was filtered through celite and the filtrate was washed with WO 01/51499 PCT/AUO1/00028 - 25 saturated NaHCO 3 solution (3 x 500 mL). The organic phase was dried over MgSO 4 and evaporated to dryness to give an oily residue. The residue was suspended in diisopropylether (150 mL) and the resulting solid was filtered. The solid 5 was washed with diisopropylether (100 mL) and dried under high vacuum at room temperature to give 3,4 methylenedioxybenzyl 4,6-0-benzylidene-2-0-(4 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-oa-D galactopyranosyl)-$-D-galactopyranoside (9) (6.7 g, 47%) as 10 a white powder. Rf 0..50 (EtOAc/.Hexane 1:1) MS (electrospray) C 62

H

55 C1 5 0 1 4 (1201.38) m/z (%)1221[M+Na]* (80). 15 3, 4-Methylenedioxybenzyl 4, 6-0-benzyli dene-3 -0- (2,3,4,6 tetra-O-(4-chlorobenzyl)-a-D-galactopyranosyl)-0-D galactopyranoside (10) To a solution of sodium methoxide (280 mg, 10.4 mmol) in dry methanol (50 mL), 3,4-methylenedioxy-benzyl 4,6-0 20 benzylidene-2-0-(4-chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4 chlorobenzyl)-ca-D-galactopyranosyl)- -D-galactopyranoside (9) (6.3 g, 5.2 mmol) in dry THF-MeOH 2:1 (150 mL) was added. The resulting mixture was stirred at 40 0 C for 5 hours. The reaction mixture was cooled to 18'C and 2.5 neutralized (pH 7.0) with Amberlite IR7120 Ht cation exchange resin. The resin waszfiltered of.f and the filtrate evaporated to dryness to give an oily residue. The crude product was suspended in hexane (200 mL), which was then vigorously stirred to break up the clumps. The suspension 30 was filtered and dried in vacuum at room temperature to -give 3,4-methylenedioxybenzyl 4,6-0-benzylidene-3-0 (2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D-galactopyranosyl) - D-galactopyranoside (10) (5.2 g, 93%) as a white powder. 35 Rf 0.30 (CH 2 Cl 2 /ethanol 50:1), MS (electrospray) m/z

C

55

H

52 C1 4 0 13 (1062.83) m/z (%) 1098[M+K]* (72) WO 01/51499 PCT/AUO1/00028 - 26 3,4 -methylenedioxybenzyl 3-O- (a-D-galactopyranosyl) -/3-D galactopyranoside (11) To a suspension of Pd/C (10%) catalyst (220 mg) in a mixture of THF-EtOH-H 2 0 6:2:1 (5 mL), a solution of 3,4 5 methylenedioxybenzyl 4,6-0-benzylidene-3-0-(2,3,4,6-tetra 0-(4-chlorobenzyl)-a-D-galactopyranosyl)-3-D galactopyranoside (10) (200 mg, 0.19 mmol) in a mixture of THF-EtOH-H 2 0 6:2:1 (5 mL) was added. The resulting suspension was shaken under a positive pressure (45 PSI) of 10 hydrogen for 2.5 h. The reaction mixture was filtered through celite and the filtrate was concentrated under high vacuum at room temperature to a volume of approximately 15 mL. The resulting yellow solution was diluted with deionised water (50 mL) and neutralized (pH 7.0) with 15 excess mixed bed resin (Amberlite-MB 1). The aqueous suspension was filtered and the filtrate was evaporated to dryness under high vacuum to give the crude product as a colourless residue. The crude product was purified by chromatography using CHCl 3 -MeOH-H 2 0 5:5:1 as the mobile 20 phase to give 3,4-methylenedioxybenzyl 3-0-(x-D galactopyranosyl)-0-D-galactopyranoside (11) (72 mg, 73%). Rf 0.42 (CHCl 3 /MeOH/H 2 0 5:5:1) MS (electrospray) C 20

H

28 0 13 (476.43) m/z (%) 499[M+Na]* (38), 477[M+H)*' (72) 25 3-0- (a-D-Galactopyranosyl) -D-galactopyranose (12) A mixture of Pd(OH) 2 (20%) Pearlman's catalyst (0.7 g) and 3,4-methylenedioxybenzyl 4,6-0-benzylidene-3-0 30 (2,3,4,6-tetra-O-(4-chlorobenzyl)-oa-D-galactopyranosyl)-f D-galactopyranoside (10) (2.0 g, 1.9 mmol) in a mixture of THF-MeOH-H 2 0 4:1:1 (30 mL) was shaken under a positive pressure (60 PSI) of hydrogen overnight. The reaction mixture was filtered through celite and the filtrate was 35 neutralized with mixed-bed ion exchange resin (Amberlite-MB 1) /negative silver (I) nitrate test/. The reaction mixture WO 01/51499 PCT/AUO1/00028 - 27 was filtered and the filtrate was concentrated to dryness in vacuum at room temperature. The residue was taken up in deionised water (2 mL) and passed through a C18 Sep Pak cartridge eluting with milli-Q-water (30 mL). The filtrate 5 was evaporated under reduced pressure to give 3-0-(x-D galactopyranosyl)-D-galactopyranose (12) (560 mg, 86%) as a white solid foam.. TLC (CHCl 3 -MeOH-H 2 0 10 : 10 : 2) Rf =0.3, High performance anion exhange chromatography with pulsed 10 amperometric detection /HPAE-PAD/ (4 x 250 mm Dionex CarbopaK-PA1 analytical column with guard column, 150 mM sodium hydroxide at 1 mL/min.) tR =5.0 min., MS (electrospray) m/z 365 [M + Na]. Rf 0.30 (CHC1 3 /MeOH/H 2 0 5:5:1) MS (electrospray) 15 C 12

H

22 0 11 (342.29) m/z (%) 406[M+Na+MeCN]* (100), 365[M+Na]* (62) Example 4: Preparation of 2-[2-(2-thiobiphenylcarbonyl) ethoxy] ethyl 3 -O-a-D-galactopyranosyl -/-D 20 galactopyranoside (23) The synthesis of the reagents for this preparation and the preparation scheme itself are set out in Reaction Schemes 2 and 3 respectively.

WO 01/51499 PCT/AUO1/00028 - 28 Reagents for the Synthesis of 2-[2-(2-Thiobiphenyl carbonyl) -ethoxyl ethyl 3-O-a-D-Galactopyranosyl-p-D Galactopyranoside 0 0 OH OH O O O 0 HO SMe HO SMe MDBnO SMe OH OH OMDBn 1 17 18 CIBnO OBnCI ClBnO %BSMe OBnGI 8 HO' -0-O ' -Cl HO -0 l-O *' S 0 13 14 HO Cl o 0 0 0 5 15 16 Reaction Scheme 2 WO 01/51499 PCT/AUO1/00028 29 QXO HO MeO HO Oz O - 0~C 0 6 19 Q O MDBnOOMDsMe HO OO M~ nO MD~O OO n 0 28 0 ~00 OBZCI0 zI OH OH IOH HO O HO OH OOHO O~ 0 O0 22 S 23 eS Reaction Scheme 3 5 2-[2-(2--Thiobenzoyl)ethoxy]ethanol (14) A mixture o f 2- [2- (2 -chloroe thoxy) ethoxy] ethanol (13 ) (17.1 g, 101 mmol) and cesium thiobenzoate (38.24 g, 142 mmol) in dry DMF (200 ml) was stirred at 751C for 1.5 hours. The reaction mixture was cooled to room WO 01/51499 PCT/AUO1/00028 - 30 temperature and evaporated to dryness. The residue was taken up in diethylether (600 ml), washed three times with saturated NaHCO 3 solution (400 ml) and with water (500 ml). The organic phase was dried over MgSO 4 and evaporated to 5 dryness to give 23 g of crude product. The crude residue was purified by chromatography using diethylether as the mobile phase to give 2-[2-(2-thiobenzoyl)ethoxy]ethanol (14) (18.75 g, 68%) as an orange syrup. 10 Rf 0.60 (diethylether/EtOH 19:1) MS (electrospray) C 13

H

18 0 4 S (270.34) m/z (%) 293[M+Na]+ (62), 271[M+H] (100) 3,4-Me thylenedioxybenzyl chloride (16) A solution of 3,4-methylenedioxybenzyl alcohol (15) 15 (50 g, 328.62 mmol) in CH 2 C1 2 (50 ml) was cooled to 0 0 C and SOCl 2 (250 ml) added dropwise. The reaction mixture was stirred at 0 0 C for 1 hour, at room temperature for 4 hours, then evaporated to dryness. The residue was purified by distillation under vacuum to give 3,4-methylenedioxybenzyl, 20 chloride (16) (49 g, 87%). Rf 0.75 (CHC 3 /EtOAc 20:1) Methyl 4, 6-0-benzylidene-l-thio-#3-D-galactopyranoside (17) 25 A mixture of methyl 1-thio--D-galactopyranoside (1) (23.6 g, 112 mmol), a,a.-dimethoxytoluene (25.62 g, 168 mmol) and p-toluenesulphonic acid (100 mg) in MeCN (500 ml) was stirred at room temperature for 30 minutes. The reaction mixture was neutralized with triethylamine 30 (1 ml) and evaporated to dryness, followed by a co evaporation with toluene. The residue was taken up in

CH

2 C1 2 (250 ml), washed twice with brine (250 ml), dried over MgSO 4 and evaporated. The resulting white solid was crystallized from EtOH to give methyl 4,6-0-benzylidene-l 35 thio-Q-D-galactopyranoside (17) (27.5 g, 82%).

WO 01/51499 PCT/AUO1/00028 - 31 Rf 0.32 (EtOAc) MS (electrospray) C 14 Hi 8 0 5 S (298.36) m/z (%) 321[M+Na]* (32), 299[M+H]* (100) Methyl 4, 6-0-benzylidene-2, 3-di-O- (3, 4-methyl enedioxy 5 benzyl)-l-thio-p-D-galactopyranoside (18) A mixture of methyl 4,6-0-benzylidene-1-thio-p-D galactopyranoside (17) (20 g, 66.80 mmol) and sodium hydride (95%) (4.80 g, 201.2 mmol) in dry DMF (350 ml) was stirred at 0 0 C for 30 minutes, then 3,4-methylenedioxy 10 benzyl chloride (34.3 g, 201,2 mmol) (16) added in DMF (20 ml). The reaction mixture was stirred at room temperature overnight. Methanol (20 ml) was added and the reaction mixture was evaporated to dryness. The residue was taken up in CH 2 Cl 2 (500 ml), washed twice with brine 15 (500 ml), dried over MgSO 4 and evaporated. The residue was crystallized from 2-propanol (1 1) to give methyl 4,6-0 benzylidene-2,3-di-0-(3,4-methylenedioxybenzyl)-l-thio-$-D galactopyranoside (18) (19 g, 50%). 20 Rf 0.62 (CHCl 3 /EtOAc 20:1), MS (electrospray) C 30

H

30 0 9 S (566.62) m/z (%) 589[M+Na]* (100), 567[M+H]* (25) 2-[2- (2-Thiobenzoyl) ethoxylethyl 4, 6-O-benzylidene 2-0- (4 chlorobenzoyl) -$-D-galactopyranosic7e (19) 25 A mixture of methyl 4,6-0-benzylidene-2-0-(4 chlorobenzoyl)-l-thio-Q-D-galactopyranoside (6) (10 g, 22.93 mmol), 2-[2-(2-thiobenzoyl)ethoxy]ethanol (13) (6.81 g, 25.22 mmol), powdered molecular sieves 4A (20 g) and dimethyl(methylthio)sulfonium tetrafluoroborate (7.0 g, 30 35.71 mmol) was stirred in dry 1,2-dichloroethane (100 mL) at 0 0 C for 2 hours. The mixture was neutralized with triethylamine (10 mL), diluted with CH 2 C1 2 (300 mL) and filtered through celite. The filtrate was washed three times with saturated sodium bicarbonate solution (200 mL), 35 dried over MgSO 4 and evaporated to dryness. The residue was suspended in diisopropylether (600 mL) and filtered. The resulting solid was crystallized from ethanol (50 ml), WO 01/51499 PCT/AUO1/00028 - 32 washed with diisopropylether (200 mL) and dried to give 2-[2-(2-thiobenzoyl)ethoxylethyl 4,6-0-benzylidene 2-0-(4 chlorobenzoyl)-$-D-galactopyranoside (19) (10 g, 66%) as a white powder. 5 Rf 0.30 (Diethylether/EtOAc 2:1), MS (electrospray)

C

33

H

35 C10 10 S (659.15) m/z (%) 681[M+Na] (70), 659[M+H1* (40) 2-[2-(2-Thiobenzoyl) ethoxy] ethyl 4, 6-O-benzylidene-2-0- (4 10 chlorobenzoyl)-3-0-[4,6-0-benzylidene-2,3-di-0-(3,4 methylenedioxybenzyl) ] -a-D-galactopyranosyl) -#-D galactopyranoside (20) A mixture of 2-[2-(2-thiobenzoyl)ethoxy]ethyl 4,6-0 benzylidene 2-0-(4-chlorobenzoyl)-0-D-galactopyranoside 15 (19) (8.55 g, 12.99 mmol), methyl 4,6-0-benzylidene-2,3-di 0--(3,4-methylenedioxybenzyl)-l-thio-$-D-galactopyranoside (18) (8.00 g, 14.29 mmol), powdered molecular sieves 4A (20 g) and methyl trifluoromethanesulfonate (4.68 g, 28.58 mmol) was stirred in dry 1,2-dichloroethane (100 mL) at 20 room temperature for 2 hours. The mixture was neutralized with triethylamine (4 mL), diluted with CH 2 Cl 2 (200 mL) and filtered through celite. The filtrate was washed three times with saturated NaHCO 3 solution (200 mL), dried over MgSO 4 and evaporated to dryness. The residue was purified 25 by chromatography using diethylether-EtOAc 2:1 as the mobile phase to give 7.5 g of 2-[2-(2 thiobenzoyl)ethoxy]ethyl 4,6-0-benzylidene-2-0-(4 chlorobenzoyl)-3-0-[4,6-0-benzylidene-2,3-di-0-(3,4 methylenedioxybenzyl)]- -D-galactopyranosyl)--D 30 galactopyranoside (20) (7.5 g, 50%) as a white solid foam. Rf 0.55 (Diethylether/EtOAc 2:1), MS (electrospray) C6 2

H

61 C1O 19 S (1177.67) m/z (%) 1199[M+Na]* (100), 1177 (21) WO 01/51499 PCT/AUO1/00028 - 33 2- [2- (2-Thiobenzoyl) ethoxy] ethyl 4, 6-0-benzylidene-2-0- (4 chlorobenzoyl)-3-0-(4,6-0-benzylidene-a-D galactopyranosyl) -#-D-galactopyranoside (21) A mixture of 2-[2-(2-thiobenzoyl)ethoxy]ethyl 4,6-0 5 benzylidene-2-0-(4-chlorobenzoyl)-3-0-[4,6-0-benzylidene 2,3-di-O-(3,4-methylenedioxybenzyl)]-a-D-galactopyranosyl) $-D-galactopyranoside (20) (7.02 g, 5.97 mmol) and 2,3 dichloro-5,6-dicyano-1,4-benzoquinone (2.71 g, 11.93 mmol) in the mixture of CH 2 Cl 2

/H

2 0 7:2 (70 ml) was stirred at room 10 temperature for 1 hour. The reaction mixture was filtered, the filtrate was diluted with CHCl 3 (300 ml), washed twice with saturated NaHCO 3 solution (150 ml) and concentrated to dryness. The residue was taken up in hot diisopropylether (150 ml) and the solution was stirred at room temperature 15 for 2 hours. The resulting suspension was filtered, then crystallized from EtOAc (40 ml). The mother liquid was purified by chromatography using diethylether-EtOAc 1:1 mixture as the mobile phase. The purified products were combined to give 2-[2-(2-thiobenzoyl)ethoxylethyl 4,6-0 20 benzylidene-2-0-(4-chlorobenzoyl)-3-0-(4,6-0-benzylidene-X D-galacto-pyranosyl)-j-D-galactopyranoside (21) (3.69 g, 68%). Rf 0.32 (Diethylether/EtOAc 2:1), MS (electrospray) 25 C 46

H

49 C10 15 S (909.40) m/z (%) 931[M+NaJ* (35), 909[M+H]* (100) 2-[2-(2-Thiobenzoyl)ethoxy]ethyl 2-0-(4-chlorobenzoyl)-3-0 a-D-galactopyranosyl -#-D-galactopyranosi de (22) 30 A mixture of 2-[2-(2-thiobenzoyl)ethoxy]ethyl 4,6-0 benzylidene-2-0-(4-chlorobenzoyl)-3-0-(4,6-0-benzylidene-a D-galactopyranosyl)-f-D-galactopyranoside (21) (3.5 g, 3.85 mmol) and p-toluenesulphonic acid (100 mg) in the mixture of acetonitrile-methanol 1:1 (350 ml) was stirred under 35 reflux for 2 hours. The reaction mixture was evaporated to dryness then the residue was chromatographed using MeCN-H 2 0 10:1 as the mobile phase to give 2-[2-(2- WO 01/51499 PCT/AUO1/00028 - 34 thiobenzoyl)ethoxy]ethyl 2-0-(4-chlorobenzoyl)-3-0-aX-D galactopyranosyl- -D-galactopyranoside (22) (2.46 g, 87%). Rt 0.42 (MeCN/H 2 0 10:1), MS (electrospray) C 32

H

41 C10 1 5 S 5 (733.13) m/z (%) 755[M+Na]* (52), 733[M+H)] (100) 2-[2-(2-Thiobiphenylcarbonyl)ethoxy]ethyl 3-O-a-D galactopyranosyl-$-D-galactopyranoside (23) A mixture of 2-[2-(2-thiobenzoyl)ethoxy]ethyl 2-0-(4 10 chlorobenzoyl)-3-0-X-D-galactopyranosyl-$-D galactopyranoside (22) (210 mg, 0.287 .mmol) and sodium methoxide (9 mg, 0.287 mmol) in dry methanol (15 ml) was stirred at 40 0 C for 4 hours. The reaction mixture was cooled to room temperature and biphenylcarbonyl chloride 15 (62.17 mg, 0.287 mmol) was added. After 30 minutes stirring at room temperature, the reaction mixture was evaporated to dryness. The residue was purified by chromatography using MeCN-H 2 0 5:1 as the mobile phase to give 2-[2-(2 thiobiphenylcarbonyl)ethoxy]ethyl 3-0-a-D-galactopyranosyl 20 $-D-galactopyranoside (23) (120 mg, 62%). Rf 0.35 (MeCN/H 2 0 10:2), MS (electrospray) C 31

H

42 0 14 S (670.73) m/z (%) 693[M+Na]* (100), 671[M+H]+ (20) 25 _Example 5: Preparation of 2-Acetamido-2-Deoxy-4-0-[3-O-(a D-Galactopyranosyl)--0-D-Galact opyranosyl] -D Glucopyranose (28) WO 01/51499 PCT/AUO1/00028 - 35 The general strategy for this preparation is set out in Reaction Scheme 4. CIBnO OBnCI Q O O CIBnO OBnCI 0 ClBnO O SMe 0 O OBnCI O O8 ClBnO O HO SMe CIBnO SMe OBzCI OBzCI 6 24 AcHN OBn BnO1~Z1 OBn 0 25 CIBnO OBnCI 0

L

0 0 AcNOn CIBnO OBnCI 00 CBnO OCnO BnO 0 AcHN OBn OH -<OCIBnO zC~ OBn OBzCI 27 OBn 26 OH OH OHOH OHHN OHH OH 5 28 Reaction Scheme 4 Methyl 4,6-0-benzylicene-2-0-(4-chlorobenzoyl)-3-0 10 (2,3, 4, 6-tetra -0- (4-chlorobenzyl ) -a-D-gal actopyranosyl) -1 thio-Q-D-galactopyranoside (24) A mixture of methyl 2,3,4, 6-tetra-O- (4-chlorobenzyl) thio-$-D-galactopyranoside (8) (3.9 g, 5.5 mmol), molecular sieves 4A (4 g) in dry THF (30 ml) was stirred at room 15 temperature, then a solution of bromine (1.18 g , 6.66 mmol) in CH 2 C1 2 (5 ml) was added. The reaction mixture was stirred at room temperature for 10 minutes, then cyclohexene (1 ml) added. To the stirred reaction mixture WO 01/51499 PCT/AUO1/00028 - 36 methyl 4,6-0-benzylidene 2-0-(4-chlorobenzoyl)-$-D galactopyranoside (6) (2.0 g, 3.7 mmol)was added then the suspension was cooled to -15 0 C. A solution of silver trifluoromethanesulphonate (1.4 g, 5.5 mmol) in dry THF (10 5 ml) was added dropwise under nitrogen atmosphere in 15 minutes. The reaction mixture was kept at 0 0 C overnight. The reaction mixture was neutralized with triethylamine (2 ml) and filtered. The filtrate was evaporated to dryness and the residue was taken up in CHCl 3 (300 mL). The 10 solution was washed with saturated NaHCO 3 solution (3 x 300 mL) . The organic phase was dried over MgSO 4 and evaporated to dryness to give an oily residue. The residue was chromatographed using diethylether-ethanol 20:1 as the mobile phase to give methyl 4,6-0-benzylidene-2-0-(4 15 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chloro-benzyl)-a'-D galactopyranosyl)-l-thio-$-D-galactopyranoside (24) (1.60 g, 40%). Rf 0.30 (Diethylether), MS (electrospray) C 55

H

51 C1 5 0 11 S 20 (1097.33) m/z (%) 1117[M+Na]* (100), 1095[M+H]* (32) Benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-0-[4,6-0 benzylidene-2-O- (4-chlorobenzoyl) -3-0- (2,3,4, 6-tetra -0-(4 chlorobenzyl) -c-D-galactopyranosyl) -0-D-galactopyranosyl) ] 25 a-D-glucopyranoside (26) A mixture of methyl 4,6-0-benzylidene-2-0-(4 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-cx-D galactopyranosyl)-1-thio- -D-galactopyranoside (24) (430 mg, 0.39 mmol), benzyl 2-acetamido-3,6-di-0-benzyl-2 30 deoxy-x-D-glucopyranoside (25) (300 mg, 0.59 mmol), molecular sieves 4A (5 g) and methyl trifluoromethane sulphonate (97 mg, 0.59 mmol) in dry 1,2-dichloroethane (15 ml) was stirred at room temperature overnight. The reaction mixture was neutralized with triethylamine (2 ml) 35 and filtered. The filtrate was diluted with CHCl 3 (100 ml) and was washed with saturated NaHCO 3 solution (2 x 100 mL). The organic phase was dried over MgSO 4 and evaporated to WO 01/51499 PCT/AUO1/00028 - 37 dryness to give an oily residue. The residue was chromatographed using diethylether-ethanol 25:1 as the mobile phase to give benzyl 2-acetamido-3,6-di-0-benzyl-2 deoxy-4-0-[4,6-0-benzylidene-2-0-(4-chlorobenzoyl)-3-0 5 (2,3,4,6-tetra-O-(4-chlorobenzyl)-a.-D-galactopyranosyl)-$ D-galactopyranosyl)]-c-D-glucopyranoside (26) (300 mg, 50%). Rf 0.33 (Diethylether/EtOH 25:1), MS (electrospray) 10 C 83

H

80 C1 5

NO

17 (1540.83) m/z (%) 1560[M+Na]* (100), 1538[M+H]* (27) Benzyl 2-acetamido-3,6-di-0-benzyl-2-deoxy-4-0-[4,6-0 benzylidene-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D 15 galactopyranosyl) -Q-D-galactopyranosyl) ] -a-D glucopyranoside (27) To a solution of sodium methoxide (73 mg, 0.13 mmol) in dry methanol (10 mL), benzyl 2-acetamido-3,6-di-0 benzyl-2-deoxy-4-0-[4,6-0-benzylidene-2-0-(4 20 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-ca-D galactopyranosyl)-$-D-galactopyranosyl)]-ca-D glucopyranoside (26) (300 mg, 0.19 mmol was added. The resulting mixture was stirred at 40'C for 4.5 hours. The reaction mixture was kept at 0 0 C for 1 hour and filtered. 25 The solid precipitate was washed with cold dry MeOH (10 ml) to give benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-0 [4,6-0-benzylidene-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a D-galactopyranosyl)-$-D-galactopyranosyl)]-a-D glucopyranoside (27) ( 190 mg, 67%) as a white powder. 30 Rf 0.35 (CHC1 3 /MeOH 7:3), MS (electrospray) C 76

H

77 ClN03i 6 (1402.27) m/z (%) 1423[M+Na]* (100), 1401[M+H]*+ (35) 2-Acetamido-2-deoxy-4-0- [3-0- (a-D-galactopyranosy1) -#-D 35 galactopyranosyl]-D-glucopyranose (28) To a suspension of Pd/C (10%) catalyst (1.0 g), benzyl 2-acetamido-3,6-di-0-benzyl-2-deoxy-4-0-[4,6-0- WO 01/51499 PCT/AUO1/00028 - 38 benzylidene-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-cL-D galactopyranosyl)--D-galactopyranosyl)]-cX-D glucopyranoside (27) (190 mg, 0.13 mmol) and acetic acid (3 drops) was shaken under a positive pressure (45 PSI) of 5 hydrogen for 4 hours. The reaction mixture was filtered through celite and the filtrate was neutralized (pH 7.0) with excess mixed bed resin (Amberlite-MB 1).The resin was filtered off and the filtrate was evaporated to dryness. The residue was taken up in milli-Q water (10 mL) and the 10 resulting solution was filtered using a 0.22 pm filter. The filtrate was passed through a C-18 Sep-pak cartridge (1 g). The filtrate was evaporated to dryness and the remaining solid was further dried over phosphorus pentoxide at room temperature under high vacuum to give 2-acetamido-2-deoxy 15 4-0-[3-0-(c-D-galactopyranosyl)- -D-galactopyranosyl]-D glucopyranose (28) (32 mg, 43%) as a white solid. Rf 0.36 (CHCl 3 /MeOH/H 2 0 10:12:3), MS (electrospray) C 20

H

35 NOi 6 (545.50) m/z (%) 568[M+Na]* (100), 546[M+H]* (52) 20 Example 6: Alternative Synthesis of Compound (28) Compound (28) may also be prepared using a different glucosamine acceptor, benzyl-6-0-benzoyl-3-0-benzoyl 1-2 acetamido-2-deoxy-x-D-glucopyranoside, using the strategy 25 set out in Reaction Scheme 5. The acceptor can readily be prepared in high yield.

WO 01/51499 PCT/AUO1/00028 - 39 OH OH O -H H Nli3gr NcOHACNHOBn NH~c 30 29 H OH 0 Ph-"Z Ph\ BnO BnOH ACNH OBn AcNHOBn AcN4HOBn 3332 31 1Ph Ph OBz 00 H 0 0 00 0 BOCIAcO SMe H SB2 e ACNHOBn OBzCl B2 3435 6 Ph Ph 0 T 0 AcNH OBII 0 Ac H OBn Olc BnO 00 BnO B2IOBz OBzC1 36 37OBz Ph CflnO OBnfI 0 0In 8 AcNH OBn CIBn 0 BnO Ph OBC1 OBz CIBn OBnCl 3 0 0 AcNH OBn CI~n Cln 0 0 BnO )I 28 OH 39 Reaction Scheme 5 WO 01/51499 PCT/AUO1/00028 - 40 2-Acetamido-2-deoxy-D-glucopyranose (29) Sodium (23.4g, 1.02 mol) was reacted with dry methanol (1.6 L), then the resulting solution was cooled to 40 'C. Glucosamine hydrochloride (200 g, 0.926 mol) was 5 added to the solution and the reaction mixture was stirred vigorously for 5 minutes. The suspension was filtered in dry conditions. Acetic anhydride (140 mL, 1.48 mol) was added dropwise to the filtrate at 0 'C in 30 min. The resulting suspension was stirred at room temperature for 10 another 30 minutes. The reaction mixture was diluted with ether (2 L), filtered and the solid product was dried to give 2-acetamido-2-deoxy-D-glucopyranose (29)(177 g, 86 %). Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside (30) 15 A mixture of 2-acetamido-2-deoxy-D-glucopyranose (29) (150 g, 0.68 mol), Amberlite IR 120 [IH] ion exchange resin (150 g) in benzyl alcohol (1.25 L) was stirred at 80 'C for 3.5 hours. The reaction mixture was filtered. The filtrate was evaporated under reduced pressure at 90 Co. The residue 20 was taken up in hot isopropanol (600 mL) and filtered. The filtrate was left to crystallize, the white crystalline solid was filtered off, washed twice with cold isopropanol (200 mL) and twice with ether (200 mL) to give 2-acetamido 2-deoxy-ax-D-glucopyranoside (30) (56.2 g, 27%). 25 Benzyl 4, 6-0-benzylidene-2-acetamido-2 -deoxy-a-D glucopyranoside (31) Benzyl 2-acetamido-2-deoxy-X-D-glucopyranoside (30) (50 g, 0.16 mmol) was dissolved in dry DMF (200 mL). Dry 30 acetonitrile (100 mL), ax,a-dimethoxytoluene (29 g, 0.19 mol, 1.2 eq) and p-toluenesulphonic acid (50 mg) was added to the DMF solution. The reaction mixture was stirred at 80 'C for 2 hours under vacuum (350 mbar); the product started to precipitate after 1 hour. The resulting suspension was 35 cooled (60 0 C) and the pH adjusted to 7 by addition of triethylamine. The suspension was cooled to 0 C, and cold methanol (500 mL) (-10 'C) was added slowly to the mixture.

WO 01/51499 PCT/AUO1/00028 - 41 The product was filtered, washed with cold methanol (200 mL) then with cold ether (2 x 200 mL) to give benzyl 4,6-0 benzylidene-2-acetamido-2-deoxy-a-D-glucopyranoside (31) (48 g, 75 %) 5 Benzyl 3-O-benzyl-4, 6-O-benzylidene-2-acetamido-2-deoxy-a D-glucopyranoside (32) A suspension of sodium hydride (3.6 g, 0.15 mol, 1.2 eq) in dry DMF (25 mL) was cooled to 0 0 C, and a solution 10 of benzyl 4,6-0-benzylidene-2-acetamido-2-deoxy-x-D glucopyranoside (32) (50 g, 0.125 mol) in dry DMF (450 mL) was added dropwise in 30 -minutes. The resulting solution was stirred at 0 0 C for 30 minutes and benzyl bromide was added (25.66 g, 0.15 mol, 1.2 eq) dropwise at 0 0 C (the 15 product started to precipitate at the beginning of the addition of -the benzyl bromide). The reaction mixture was stirred at room temperature for 45 minutes, cooled to 0 'C and dry methanol (25 mL) was added dropwise. The reaction mixture was diluted with cold ether (1 L) and the mixture 20 was stirred for 30 minutes. The resulting suspension was filtered and washed three times with ether (400 mL) to give benzyl 3-0-benzyl-4,6-0-benzylidene-2-acetamido-2-deoxy-a D-glucopyranoside (32) (62.0 g) as a white powder with quantitative yield. 25 Benzyl 3 -O-benzyl -2-ace tamido-2-cdeoxy-a-D-glucopyranoside (33) A suspension of benzyl 3-0-benzyl-4,6-0-benzylidene 2-acetamido-2-deoxy-a-D-glucopyranoside (32) (50 g, 0.102 30 mol) in acetic acid (500 mL) and water (25 mL) was stirred at 110 0 C for 45 minutes. The reaction mixture was concentrated under reduced pressure at 40 C'. The oily residue was taken up twice in toluene (200 mL) and concentrated. The residue was treated with di-isopropyl 35 ether (250 mL) and the resulting suspension was strirred for 30 minutes. The white solid was filtered off, washed twice with cold ether (200 mL) to give benzyl 3-0-benzyl-2- WO 01/51499 PCT/AUO1/00028 - 42 acetamido-2-deoxy-aX-D-glucopyranoside (33) (38.0 g, 93%). Benzyl 6-0-benz oyl-3 -0-benzy1 -2-ace tamido-2-d eoxy-a-D glucopyranoside (34) 5 A solution of benzoyl chloride (6.3g, 0.045 mol, 1.2eq) and imidazole (6 g, 0.09mol, 2.4 eq) in dry 1,2 dichloroethane (150 mL) was stirred for 20 minutes at 5 'C. The resulting suspension was filtered under dry conditions. The filtrate was added to a solution of benzyl 3-0-benzyl 10 2-acetamido-2-deoxy-a-D-glucopyranoside (33) (15g, 37.6 mmol) in dry 1,2-dichloroethane (600 mL). The reaction mixture was stirred at 90 0 C for 48 hours and cooled to room temperature. The resulting suspension was filtered, washed twice with brine (300 mL), dried over MgSO 4 and 15 concentrated. The residue was taken up in hot isopropanol (300 mL) and left to crystallize. The white crystalline solid was filtered off to give Benzyl 6-0-benzoyl-3-O benzyl-2-acetamido-2-deoxy-a-D-glucopyranoside (34) (11.7 g, 62%). 20 Methyl 4,6-0-benzylidene-3-0-chloroacetyl-2-0-(4 chlo.robenzoyl) -l-thio-Q-D-galactopyranoside (35) A mixture of methyl 4,6-0-benzylidene-2-O-(4 chlorobenzoyl)-l-thio- -D-galactopyranoside (6) (10.0 g, 23 25 mmol) and 4-dimethylaminopyridine (3.40 g, 27.8 mmol) in dry 1,2-dichloroethane (100 mL) was stirred at 0 'C, then chloroacetyl chloride (3.4 g , 27.8 mmol, 1.2 eq) was added dropwise to the solution. The reaction mixture was stirred at room temperature for 2.5 hours, then diluted with 1,2 30 dichloroethane (100 mL). The resulting solution was washed twice with saturated brine solution (100 ml), dried over MgSO 4 and concentrated to give methyl 4,6-O-benzylidene-3 0-chloroacetyl-2-0-(4-chlorobenzoyl)-1-thio- -D galactopyranoside (35) (10.2 g, 86%) as a white crystalline 35 solid.

WO 01/51499 PCT/AUO1/00028 - 43 Benzyl 2-ace tamido-6-0-benzoyl -3-0-benzyl -4-0- [4, 6-0 benzyl idene-3-0-chloroacetyl-2-0- (4-chlorobenzoyl) -J3-D galactopyranosyl ]-2-deoxy-a-D-glucopyranoside (36) To a mixture of benzyl 2-acetamido-6-0-benzoyl-3-0 5 benzyl-4-O-2-deoxy-a-D-glucopyranoside (34) (5 g, 9.9 mmol), methyl 4,6-0-benzylidene-3-0-chloroacetyl-2-0-(4 chlorobenzoyl)-1-thio--D-galactopyranoside (35) (5.71 g, 11.1 mmol, 1.12 eq) and Molecular sieves 4A (2.5 g) in dry 1,2-dichloroethane (300 mL), DMTST (5.75g, 2.4 eq) was 10 added under nitrogen. The reaction mixture was stirred at room temperature for 5 hours, then neutralized by addition of pyridine (5 mL). Acetic anhydride was added (2.5 mL) and the reaction mixture was stirred at room temperature for 0.5 hours. The resulting suspension was filtered through a 15 bed of Celite. The filtrate was washed with a saturated solution of NaHCO 3 (200 mL), twice with brine (200 ml), dried over MgSO 4 and concentrated. The residue was taken up in DCM (25 mL) and diisopropyl ether (200 mL) was added. The resulting yellow precipitate was filtered off and 20 washed twice with cold diisopropyl ether (100 mL). The solid was crystallized using a mixture of DCM (20 mL)and ether (25 mL) to give benzyl 2-acetamido-6-0-benzoyl-3-0 benzyl-4-O-[4,6-0-benzylidene-3-0-chloroacetyl-2-0-(4 chlorobenzoyl)-$-D-galactopyranosyll-2-deoxy-a-D 25 glucopyranoside (36) (5.1 g, 55%) as a white crystalline solid. Benzyl 2-acetamido-6-0-benzoyl-3-0-benzyl-4-0-[4,6-0 benzylidene-2 -0- (4 -chlorobenzoyl) -p-D-galactopyranosyl ] -2 30 deoxy-a-D-glucopyranoside (37) A mixture of benzyl 2-acetamido-6-0-benzoyl-3-0 benzyl-4-0-[4,6-0-benzylidene-3-0-chloroacetyl-2-0-(4 chlorobenzoyl)-$-D-galactopyranosyl]-2-deoxy-a-D glucopyranoside (36) (0.5 g) and thiourea (303 mg) in THF 35 (3 mL) and water (0.5 mL) was stirred at room temperature for 14 hours, then the reaction mixture was diluted with chloroform (100 mL). The resulting solution was washed WO 01/51499 PCT/AUO1/00028 - 44 twice with water (50 ml), dried over MgSO 4 and concentrated. The residue was purified by flash chromatography using DCM / EtOAc 1:1 as the mobile phase to give benzyl 2-acetamido-6-0-benzoyl-3-0-benzyl-4-0-[4,6-0 5 benzylidene-2-0-(4-chlorobenzoyl)- -D-galactopyranosyl]-2 deoxy-aX-D-glucopyranoside (37) (280 mg, 61 %) as a white solid. Benzyl 2-acetamido-6-0-benzoyl-3-0-benzyl-2-deoxy-4-0-[4,6 10 0-benzylidene-2-0-(4-chlorobenzoyl)-3-0-(2,3,4,6-tetra-0 (4 -chl orobenzyl) -ac,3-D-galact opyranosyl) -j-D galactopyranosyl) j -a-D-glucopyranoside (38) To a mixture of methyl 2,3,4,6-tetra-O-(4 chlorobenzyl)-l-thio-$-D-galactopyranoside (430 mg, 0.602 15 mmol), benzyl 2-acetamido-6-0-benzoyl-3-0-benzyl-4-0-[4,6 0-benzylidene-2-O-(4-chlorobenzoyl)-$-D-galactopyranosyl] 2-deoxy-aX-D-glucopyranoside (37) (280 mg, 0.301 mmol) and molecular sieves 4A (300 mg) in dry 1,2-dichloroethane (3 mL), DMTST (300 mg, 1.2 mmol) was added. The reaction 20 mixture was stirred at room temperature for 3 hours. The reaction mixture was neutralized with triethylamine (1 ml), diluted with CHCl 3 (50 mL) and filtered. The filtrate was then washed with-saturated NaHCO 3 solution (3 x 50 mL). The organic phase was dried over MgSO 4 and evaporated to 25 dryness to give a solid foam. The-residue was purified by chromatography using CHCl 3 - EtOAc 1:1 as the mobile phase to give benzyl 2-acetamido-6-0-benzoyl-3-0-benzyl-2-deoxy 4-0-[4,6-0-benzylidene-2-0-(4-chlorobenzoyl)-3-0-(2,3,4,6 tetra-O-(4-chlorobenzyl)-a,$-D-galactopyranosyl)-J-D 30 galactopyranosyl)]-a-Dglucopyranoside (38) (325 mg, 70%, / = 85/15).

WO 01/51499 PCT/AUO1/00028 - 45 Benzyl 2-acetamido-3-0-benzyl-2-deoxy-4-0-[4,6-0 benzylidene-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D galactopyranosyl) -#l-D-galactopyranosyl) ] -a-D glucopyranoside (39) 5 To a solution of sodium methoxide (20 mg, 0.37 mmol) in dry methanol (2 mL),- benzyl 2-acetamido-6-0-benzoyl-3-0 benzyl-2-deoxy-4-0-[4,6-0-benzylidene-2-0-(4 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a,$-D -galactopyranosyl)- -D-galactopyranosyl)]-a-D 10 glucopyranoside (38) (190 mg, 0.12 mmol was added. The resulting mixture was stirred at 40 0 C for 4 hours. The reaction mixture was cooled to room temperature and filtered. The solid precipitate was washed with cold dry MeOH (10 mL), followed by hexane (2 x 25 mL) to give benzyl 15 2-acetamido-3-0-benzyl-2-deoxy-4-0-[4,6-0-benzylidene-3-0 (2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D-galactopyranosyl) - D-galactopyranosyl)]-a-D-glucopyranoside (39) (110 mg, 68%) as a white powder.TLC Rf 0.35 (EtOAc/CHC1 3 7:3 20 2-Acetamido-2-deoxy-4-0-[3-0-(c-D-galactopyranosyl)-03-D galactopyranosyl] -D-glucopyranose (28) To a suspension of Pd/C (10%) catalyst (100 mg), benzyl 2-acetamido-3-0-benzyl-2-deoxy-4-0-[4,6-0 benzylidene-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a,-D 25 galactopyranosyl)- -D-galactopyranosyl)]- -o.D glucopyranoside (39) (80 mg, 0.06 mmol) and acetic acid (3 drops) in THF-MeOH-H20 5:1:1 (7 mL) was shaken under a positive pressure (60 PSI) of hydrogen overnight. The reaction mixture was diluted with milliQ water (30 mL), 30 filtered through Celite and the filtrate was neutralized (pH 7.0) with excess mixed bed resin (Amberlite-MB 1).The resin was filtered off and the filtrate was evaporated to dryness. The residue was taken up in milli-Q water (5 mL) and the resulting solution was passed through a C-18 Sep 35 pak cartridge (1 g). The filtrate was evaporated to dryness and the remaining solid was further dried over phosphorus pentoxide at room temperature under high vacuum to give 2- WO 01/51499 PCT/AUO1/00028 - 46 acetamido-2-deoxy-4-0-[3-0-(C-D-galactopyranosyl)-J-D galactopyranosyl]-D-glucopyranose (28) (20 mg, 53%) as a white solid. 5 R 0.36 (CHCl 3 /MeOH/H 2 0 10:12:3), MS (electrospray) C 20

H

35

NO

1 6 (545.50) m/z (%) 568[M+Na]* (100), 546[M+H)* (52) Example 6: Immobilization of 2-acetamido-2-deoxy-4-O-[3-0 (a-D-galactopyranosyl) -/-D-galactopyranosyl] -D 10 glucopyranose (28) The following reaction scheme, Scheme 6, illustrates how a compound of the invention can be bound to a solid support, using two alternative linking groups. The second linking group is a dioxo compound, as discussed in our 15 International patent application No. PCT/AU98/00808. It will be appreciated that other compounds of the invention can be linked to a solid support in a similar manner.

WO 01/51499 PCT/AUO1/00028 - 47 OH OH OH OH HO0 OH OH H 28 AcHN OH OH HO OH HO NH 0 0 OH H NH2 40 AcHN OH OH 0O l 0 1 OH H HN 41 AcHN OH OH 0oH. OH H OOH -

O

OH H NH Ac4N OH OH OH 0 O H H, NH 42 AcHN NH-- ol 0 Scheme 6 WO 01/51499 PCT/AUO1/00028 - 48 Scheme 7: Synthesis of Participating Galactopyranoside Building Blocks O OTBDMS O OTBDMS OH OH O )SMe O SMe HO SMe OH OR OR 44 (R=Pivaloyl) 45 (R=Pivaloyl) 48 (R=Acetyl) 48 (R=Acetyl) Ph-''O Ph~ O 0 0 FmocO SMe HO SMe OR OR 43 (R=4-Chlorobenzoyl) 46 (R=Pivaloyl) 47 (R=Pivaloyl) 50 (R=Ac) Scheme 7 5 Example 7: Synthesis of Methyl 4,6-0-benzylidene-2-O-(4 chlorobenzoyl) -3-0 -fluorenylmethyl -oxycarbonyl 1-thio-/-D-galactopyranoside 10 Methyl 4,6-O-benzylidene-2-0-(4-chlorobenzoyl)-3-0-fluorenylmethyloxycarbonyl-1 thio-fp-D-galactopyranoside (43) A suspension of methyl 4,6-0-benzylidene-2-0-(4-chlorobenzoyl)-1-thio-3-D galactopyranoside 6 [20g, 45.87mmol] in 1,2-dichloroethane [200mL] was cooled to OC. To the cooled suspension was added DMAP [16.8 1g, 138mmol) followed by 15 . Fmoc-Cl [35.60g, 137mmol]]. The now solution was returned to ambient temperature and stirred for 2 hours. The reaction mixture was then diluted with Chloroform [200mL] and washed with 5% citric acid solution [2 x 400mL] and saturated brine solution [2 x 400mL]. The layers were separated and the organic layer dried over Na 2

SO

4 followed by filtration and removal of the solvent in vacuo. The resulting residue was purified by 20 column chromatography [20% ethylacetate/petroleum ethers v/v] to afford methyl 4,6 0-benzylidene-2-0-(4-chlorobenzoyl)-3-0-fluorenylmethyloxycarbonyl-1-thio- -D galactopyranoside 43 as a white foam [27.2g, 90%]; Rf = 0.22; ES-MS gave mn/z (ion, relative intensity); 'H NMR (CDC 3 ) 8 7.88-7.07 (17H, aromatic), 6.01 (t, 1H, H-2), 5.79 (s, 1H, benzylidene) 5.36 (dd, 1H, H-3), 4.91 (d, 1H, JI- 2 =8.5, H-1), 4.89 (d, 1H, H- WO 01/51499 PCT/AUO1/00028 - 49 4), 4.78 (dd, 1H, H-6a), 4.67 (in, 2H, Fluorenyl-CH 2 -), 4.52 (t, 1H, 9-fluorenyl inethyne), 4.49 (dd, 1H, H-6b), 4.14 (s, 1H, H-5), 2.29 (s, 3H, S-CH 3 ) Example 8: Synthesis of Methyl 4,6-0-benzylidene-3-0 5 fluorenylmethyloxycarbonyl-2-0-pivaloyl-l-thia B-D-galactopyranoside Methyl 6-0-tert-butyldimethylsilyl)-3,4-0-isopropylidene-2-0-(pivaloyl)-1-thio-/3-D galactopyranoside (44) 10 To a mixture 6-O-tert-butyldimethylsilyl-3,4-0 isopropylidene-l-thio-3-D-galactopyranoside [11.5g, 31.59mmoll and DMAP [5.5g, 45.5mmol] in 1,2-dichloroethane [40mL] was added dropwise, 2,2, 2-trimethylacetylchloride. The reaction was stirred for 2 hours then diluted with 15 chloroform [1OOmL) and washed with 10% citric acid solution [2 x 150mL] , saturated NaHCO 3 solution [2 x 150mL) and saturated brine solution [2 x 150mL) . The layers were separated and the organic layer dried over Na 2

SO

4 . The solvent was removed in vacuo to give an oily residue. The 20 residue was .purified by column chromatography (5%ethylacetate/petroleum ethers) to give a white foam, methyl 6-0-tert-butyldimethylsilyl-3 , 4-0-isopropylidene-2-. 0-pivaloyl-l-thio-$-D-galactopyranoside 44 [13.7g, 97%]. Rf = 0.75 (ethylacetate/petroleum ethers, 1:2, v/v); 1H NMR 25 (CDCl 3 ) 8 5.05 (dd, 1H, H-2), 4..29 (dd, 1H, H-4), 4.25 (d, 1H, J1- 2 =10.12, H-1), 4.17 (dd, 1H, H-3), 3.93-3.84 (m, 3H, H-6a, H-6b, H-5), 2.16 (s, 3H, S-CH 3 ), Methyl 2-0-pivaloyl-]-thio-/-D-galactopyranoside (45) 30 Methyl 6-0-tert-butyldimethylsilyl-2-0-pivaloyl-3, 4-0 isopropylidene-l-thio-Q-D-galactopyranoside 44 [3.34g, 7.45mmol] x, was dissolved in 25% acetonitrile/methanol [40mL]. To the solution was added 4-toluenesulphonic acid [17mg, 90.43gmol], the solution was then stirred under 35 refluxed for 3 hours. The reaction temperature was then reduced to 40'C and left overnight. The reaction mixture was then concentrated and the residue azeotroped with WO 01/51499 PCT/AUO1/00028 - 50 toluene followed by diethylether to give a white residue. The residue was purified by column chromatography (10% acetonitrile/ethylacetate, v/v) to give a white solid, methyl 2-0-pivaloyl-1-thio- -D-galactopyranoside 45 [2.19g, 5 83%], Rf = 0.20 (ethylacetate); ES-MS m/z (ion, relative intensity) .295 ([M+H] 4 , 100%); H NMR (CDCl 3 ) 85.08(dd,11,H 2), 4.39 (d, 1H, J1 2 = 9.88Hz, H-1), 4.13 (d, 1H, H-4), 4.01-3.92 (m, 2H, H-6a, H-6b), 3.72 (dd, 1H, H-3), 3.62 (dd, 1H, H-5), 2.21 (s, 3H, S-CH 3 ), 1.27 (s, 9H, t-butyl). 10 Methyl 4, 6-0-benzylidene-2-O-pivaloyl-l-thio-Q3-D galactopyranosi de (46) A mixture of methyl 2-O-(pivaloyl)-l-thio-0-D 15 galactopyranoside 45 [1.68g, 5.71mmoll, x,a dimethoxytoluene and 4-toluenesulphonic acid [10mg, 43.19mmol] was dissolved in acetonitrile [50mL] and heated at 60'C with stirring for 1 hour. The reaction was then allowed to return to ambient temperature, neutralised with, 20 2 equivalents of triethylamine and concentrated under vacuum. -The residue was taken up in chloroform [1OOmL) and, the organic layer washed with dilute brine [3:1; H 2 0:Brine,, 1.x 100mL], saturated NaHCO 3 solution [1 x 100mL], and saturated brine solution [1 x 100mL). The layers were 25 separated and the organic layer dried over Na 2

SO

4 . The organic layer was concentrated and the residue purified by column chromatography (33% ethylacetate/petroleum ethers, v/v) to give methyl.4,6-0-benzylidene-2-0-pivaloyl-1-thio P-D-galactopyranoside 46 [1.91g, 87%). Rf = 0.63 30 (ethylacetate), ES-MS m/z (ion, relative -intensity) 341 ([M+H] , 100%); 1 H NMR (CDCl 3 ) 5 7.51 (m, 2H, aromatic) 7.41 (m, 3H, aromatic), 5.58 (s, 1H, CH-benzylidene), 5.24 (dd, 1H, H-2), 4.4 (dd, 1H, H-6a), 4.39 (d, 1H, J 1 2 = 9.77, H 1), 4.29 (dd, 1H, H-4), 4.08 (dd, 1H, H-6b), 3.8 (ddd, 1H, 35 H-3), 3.60 (s, 1H, H-5),, 2.26 (s, 3H, S-CH 3 ), 1.27 (s, 9H, t-butyl) WO 01/51499 PCT/AUO1/00028 - 51 Methyl 4, 6-O-benzyli dene-3 -O-fluorenylmethyloxycarbonyl -2 O-pivaloyl-l-thio-J-D-galactopyranoside (47) Methyl 4,6-O-benzylidene-2-O-pivaloyl-l-thio-0-D galactopyranoside 46 [1.90g, 4.97mmol] was dissolved in 5 1,2-dichloroethane (20mL) and the resulting solution was cooled to 0 0 C. At this time DMAP [1.82g, 14.92mmol] and. Fmoc-Cl [3.87g, 14.92mmol] were added sequentially. The cold bath was then removed, and the reaction allowed to return to room temperature. The reaction was stirred at 10 ambient temperature for 2 hours and then diluted with CHCl 3 [~50mL]. The reaction mixture was then washed with 5% citric acid solution [2 x 1OOmL] and saturated brine solution [2 x 1OOmL]. The layers were separated and the organic layer dried over Na 2

SO

4 . The solution was then 15 filtered and concentrated to afford a yellow residue which was purified by column chromatography (20% ethylacetate/petroleum ethers v/v) to give methyl 4,6-0 benzylidene-3-0-fluorenylmethyloxycarbonyl-2-0-pivaloyl-l thio-P-D-galactopyranoside 47 [2.74g, 91%]; Rf = 0.38 (25% 20 ethylacetate/petroleum ethers v/v); ES-MS m/z (ion, intensity); 1H NMR (CDCl 3 ) 8 7.78-7.25 (13H, aromatic), 5.61 (t, lH, H-2), 5.57 (s, 1H, benzylidene), 4.97 (dd, 1H, H-3), 4.50 (d, 1H, H-4), 4.45 (d, 1H, Ji- 2 =9.10hz, H-1), 4.47-4.33 (m, 2H, Fmoc-CH 2 -), 4.25 (t, 1H, 9-fluorenyl 25 methyne), 4.40, (dd, 1H, H-6a) 4.08 ('dd, 1H, H-6b) 3.65 (s, 1H, H-5), 2.30 (s, 3H, S-CH 3 ), 1.20 (s, 9H, t-butyl) WO 01/51499 PCT/AUO1/00028 - 52 Example 9: Synthesis of Synthesis of methyl 2-0-acetyl 4,6-0-benzylidene-3-0 fl uorenylme thyl oxycarbonyl -1-thi o-Q-D galac topyranoside 5 Synthesis of .methyl 2-O-acetyl-6-O-tert-butyldimethylsilyl 3,4-0-isopropylidene-l-thio-#-D-galactopyranoside (48) A mixture of methyl 6-0-tert-butyldimethylsilyl-3,4 .O-isopropylidene-l-thio- -D-galactopyranoside (3.00g, 10 8.24mmol) and 4-dimethylaminopyridine (2.42g, 19.78mmol) in dry 1,2-dichloroethane (750 ml) was stirred at room temperature. Acetyl chloride [1.05mL, 14.84mmol] was added dropwise to the solution over 15 minutes. The reaction stirred at -room temperature for 2 hours at-which time it 15 was diluted with chloroform and washed with 10% citric acid solution [2 x 1OOmL] saturated sodium hydrogen carbonate [2 x 1OOmL]. and finally with saturated brine solution [2 x 100mL]. The layers were separated and the organic layer dried over Na 2

SO

4 . The solution was filtered and 20 concentrated to afford a white residue which was purified by column chromatography (20% ethylacetate/petroleum ethers v/v) to afford methyl 2-0-acetyl-6-0-tert butyldimethylsilyl-3,4-0-isopropylidene-l-thio- -D galactopyranoside 48 as a white solid [2.65g, 79%]; Rf 25 0.43 (25% ethylacetate/petroleum ethers v/v) Synthesis of methyl 2-0-acetyl1--thio-#3-D-galactopyranoside (49) 2-0-Acetyl-6-0-tert-butyldimethylsilyl-3,4-0 30 isopropylidene-l-thio- -D-galactopyranoside x was dissolved in 50% -acetonitrile/methanol [5OmL] and heat-ed at 60'C. To the stirred solution was added 4-toluenesulphonic acid [I10.mg, 53.19pmol] and the reaction was left for 4 hours. The reaction temperature was then reduced to 40'C and left 35 overnight. The reaction mixture was then concentrated and the residue crystallised from methanol to afford 2-0 acetyl-l-thio-]-D-galactopyranoside 49 as a white solid WO 01/51499 PCT/AUO1/00028 - 53 [1.26g, 79%3; Rf = 0.2 (25% acetonitrile/ethylacetate, v/v); 'H NMR (d-MeOH) 8 3.95 (t, 1H, H-2), 3.27 (d, 1H, Ji 2=8.63, H-1), 2.92, 1H, H-4), 2.79-2.69 (m, 2H, H-6a and H 6b), 2.62 (t, 1H, H-3), 2.38 (m, 1H, H-5) 1.37 (s, 3H, S 5 CH 3 ), 1.31 (s, 3H, -C(O)CH 3 ) Synthesis of methyl 2-0-acetyl-4,6-0-benzylidene-3-0 fluorenylmethyloxycarbonyl-l-thio-#-D-galactopyranoside (50) 10 2-0-Acetyl-1-thio- -D-galactopyranoside 49 was dissolved in acetonitrile [20mL] and heated to 60'C. To the stirred solution was added aC-dimethoxytoluene [1.09g, 7.10mmol] and 4-toluenesulphonic acid [10mg, 53.19gmol]. The reaction was stirred for 2 hours and then allowed to return to room temperature. The reaction was neutralised with 2 equivalents of triethylamine and evaporated to dryness. The residue 15 was taken up in chloroform and washed with dilute brine [1 x lOOmL], saturated sodium hydrogencarbonate solution [1 x lOOmL] and saturated brine solution [1 x 100mL]. The layers were separated and the organic layer dried over Na 2

SO

4 . The solution was filtered and concentrated. The residue was washed successively with petroleum ethers, and the resulting white solid then suspended in toluene and any remaining water azeotroped 20 under co-evaporation. The residue from the previous step was suspended in 1,2 dichloroethane [20mL] and cooled to 0 0 C. To the stirred suspension at 0C was added 4,4-dimethylaminopyridine [1.62g, 13.23mmol] and Fmoc-Cl [3.42g, 12.23mmol]. The now solution was allowed to return to room temperature and stirred for 1 hour. At this time the reaction was diluted with chloroform and washed with 5% citric acid solution 25 [2 x 75mL] and saturated brine solution [2 x 75mL]. The layers were then separated and the organic layer dried over Na 2

SO

4 . The solution was filtered and the solvent removed in vacuo to give a yellow oily residue which was purified by column chromatography (33% ethylacetate/petroleum ethers v/v) to give methyl 2-0-acetyl-4,6-0-benzylidene-3 0-fluorenylmethyloxycarbonyl-1-thio-D-D-galactopyranoside 50 [2.19g, 82%] Rf = 0.2 30 (33% ethylacetate/petroleum ethers, v/v); 1H NMR (CDCl 3 ) 8 7.78-7.24 (13H, aromatic), 5.60 (t, 1H, H-2), 5.55 (s, 1H, benzylidene), 4.88 (dd, 1H, H-2), 4.50 (d, 1H, H-4), 4.55-4.38 (m, 4H, H-1, Fmoc-CH 2 , H-6a), 4.28 (t, 1H, 9-fluorenyl-methyne), 4.06 (dd, 1H, H- 6 b), 3.63 (s, 1H, H-5), 2.29 (s, 3H, S-CH 3 ), 2.1 (s, 3H, -C(O)CH 3

)

WO 01/51499 PCT/AUO1/00028 - 54 Scheme 8: Solid Phase Synthesis of Galcx(1-3)-1(1-4)-GlcNAc OBn OBn OBn HO- OOBn 00~O H - 0NH BnOO BnOS \= OBn 0 ~ H

NH

2 a 51 0 H 52d 53 0 O 43 (R=p-CIBz) FmoO SMe 47 (R=Piv) FmoO O 50 (R=Ac) OR~ O~n OR OR OnFmoc OR --- HO- 4 \: .AONH 0 nO% - N 0 n NH 00 0 hJ NH j/J NH4 d 55 5 CIBnO OBnCI In nC CCI~n O~nC0 CI n SOORCIn OR~n Cn ,OOR CIBnO O NH 0BiOBn 00 0 oF n NH 2 Ph-/ -0 57 (R=Pivaloyl) NH- Ph.-/ 60 (R=H) 56 CnOOBnCI CIBnO OBnCI C n 0C Bn 0 O B n CIBnO OBn OR NH cOBn 0BNHAc 0'-0 61 Ph4J/ 58 (R=PivaloyI) Ph-// 59 (R=4-Chlorobenzoyl) WO 01/51499 PCT/AUO1/00028 - 55 Example 10: Synthesis of a partially protected resin linker-sugar conjugate. 5 Benzyl 3, 6-di-O-benzyl-2-deoxy-2-amino-#-D-glucopyranoside (51) To a solution benzyl of 3,6-di-O-benzyl-2-deoxy-2 phthalimido-3-D-glucopyranoside [6.20g, 10.71mmol] in ethanol [100mL], was added hydrazine hydrate [6.2mL, 10 55%/H201 and water [5mL]. The solution was refluxed overnight and then allowed to return to ambient temperature. The solution was filtered, the solvent removed in vacuo, and the residue taken up in CHCl 3 [200mL]. The Chloroform suspension was filtered, the filtrate dried over 15 Na 2

SO

4 and concentrated under reduced pressure to give a pure clear oil, benzyl 3,6-Di-O-benzyl-2-deoxy-2-amino- -D glucopyranoside 51 [4.7g, 97%]; Rf = 0.5 (Acetonitrile), ES-MS gave m/z (ion, relative intensity): 450 ([M+H]*, 100%); 1H NMR (CDCl 3 ) 8 7.43-7.30 (m 15H, aromatic), 5.00 20 4.60 (6H, 3CH 2

-C

6

H

5 ), 4.38 (d, 1H, JI, 2 = 7.92Hz, H-1), 3.85 3.75 (m, 3H, H-6a, H-6b, H-3), 3.53 (ddd, lH, H-5), 3.38 (dd, 1H, H-3), 2.92 (dd, 1H, H-2). Benzyl 3, 6-Di-O-benzyl-2-deoxy-2-N-(6-(4, 4-dimethyl -2, 6 25 dioxocyclohexylidene)-pentanoic acid-6-yl)-Q-D glucopyranoside (52) To a solution of Benzyl 3,6-Di-O-benzyl-2-deoxy-2 amino-p-D-glucopyranoside 51 [4.70g, 10.47mmol] in ethanol [100mL], was added 6-hydroxy-6-(4,4-dimethyl-2,6 30 dioxocyclohexylidene)-pentanoic acid [5.32g, 20.93mmol] followed by the addition of triethylamine [1.5mL, 10.69mmol]. The reaction mixture was heated overnight at 60'C and then allowed to return to room temperature. The reaction mixture was concentrated and the residue taken up 35 in chloroform [200mLl. The organic layer was washed with a solution of 0.3N HCl [2 x 200mL) and saturated Brine solution [1 x 200mL]. The organic layer was dried over WO 01/51499 PCT/AUO1/00028 - 56 Na 2

SO

4 and concentrated to give a pale yellow residue. The residue was purified by column chromatography with ethylacetate-petroleum ethers-acetic acid, 5:15:0.4 to give benzyl 3,6-Di-O-benzyl-2-deoxy-2-N-(6-(4,4-dimethyl-2,6 5 dioxocyclohexylidene)-pentanoic acid-6-yl)-p-D glucopyranoside 52 [6.09g, 85%]. Rf = 0.10 (ethylacetate petroleum ethers-acetic acid, 5:15:0.4), ES-MS m/z (ion, relative intensity): 686.5 ([M+H]*, 100%) 10 Coupling of Benzyl 3, 6-Di-O-benzyl-2-deoxy-2-N-(6-(4,4 dimethyl-2, 6-dioxocyclohexylidene) -pentanoic acid-6-yl) -p D-glucopyranoside to MBHA. resin (0. 7mmol/g) (53) In a 200mL peptide reaction vessel MBHA resin [11.86g, 8.30 mmol] was swollen in a minimum of dry N,N 15 dimethylformamide (DMF). A DMF [50mL] solution was made of Benzyl 3,6-Di-O-benzyl-2-deoxy-2-N-(6-(4,4-dimethyl-2,6 dioxocyclohexylidene)-pentanoic acid-6-yl)-p-D glucopyranoside 52 [6.09g, 8.90 x mmoll, diisopropyl ethylamine (DIPEA) [3.11mL, 17.8mmol] and O-Benzotriazole 20 1-yl-N,NN', N' -tetramethyluroniumhexa-fluorophosphate (HBTU) [3.37g, 8.9mmol] which was then added to the reaction vessel. The vessel was sealed and shaken overnight. Ninhydrin assay indicated that the reaction was greater than 99.4% complete, the reaction was stopped, and 25 the resin was washed with DMF [4 x 1OOmL), 50% DCM/MeOH [4 x 100mL] and DCM [4 x 10QmL]. The resin was dried under house vacuum for 4 hours and then dried under high vacuum overnight. Yield of resin 53 was [17.15g, 98.6% by weight]. 30 Synthesis of benzyl 2-acetamido-3,6-di-Q-benzyl-2-deoxy-4 0-[4,6-0-benzylidene-2-0-pivaloyl-3-0-(2,3,4,6-tetra-O-(4 chlorobenzyl) -a-D-galactopyranosyl) -0-D-galactopyranosyl) ] /-D-gl ucopyranoside (58) Under an atmosphere of nitrogen, resin 53 [300mg, 35 141pmol], 4,6-0-benzylidene-3-0-fluorenylmethyloxycarbon yl-2-O-pivaloyl-l-thio--D-galactopyranoside 47 [557mg, 846smol] and powdered molecular sieves 4i [600mg], were WO 01/51499 PCT/AUO1/00028 - 57 suspended in dichloromethane [3mL), followed by the addition of methyl trifluoromethanesulphonate [95.7gL, 846pmol]. The reaction vessel was sealed and the reaction mixture agitated for five hours at ambient temperature. The 5 resin was then washed with DMF [3 x 20mL), 50% MeOH/DCM [3 x 20mL] and DCM [3 x 20mL]. The resin was then floated in DCM to separate the resin from any remaining sieves. Resin 54 was collected and dried under house vacuum for 1 hour. The resin was then treated with a 20% triethylamine/DMF 10 solution for 25mins followed by workup as above. Resin 55 was dried under hi-vacuum overnight. Under an atmosphere of nitrogen the resin was then combined with methyl 2,3,4,6 tetra-O-(4-chlorobenzyl)-l-thio- -D-galactopyranoside 8 [600mg, 846pmol], powdered molecular sieves 4i [800mm] and 15 dichloromethane [4mL], followed finally by the addition of methyl.-trfluoromethanesulphonate [95.74pL, 846pmol]. The reaction vessel was sealed and the reaction mixture agitated at ambient temperature for five hours. The resin was then washed as standard and collected and dried on a 20 sintered funnel. In a reaction vessel resin 56 was then combined with a 5% hydrazine hydrate(55%/H 2 0)/DMF [5mL] solution and agitated at ambient temperature for 4h. The DMF solution was filtered from the resin and the resin then further washed with DMF [7mL]. The filtrates were combined 25 and the solvent removed in vacuo. The residue was taken up in minimal dichloromethane and-passed through a plug of silica (eluent; DCM, TLC: CH 2 Cl 2 :MeOH, 20:0.3'). The combined fractions were concentrated, residue 57 was then taken up in 1,2-dichloroethane [3mL] and reacted with 30 acetylchloride [461L, 648[moll in the presence of DMAP [84mg, 684pnol] for three hours at ambient temperature. The reaction was diluted with chloroform [20mL] and washed with saturated citric acid solution [2 x 20mL], saturated sodium hydrogen carbonate solution [2 x 20mL] and saturated brine 35 solution [2 x 20mL]. The organic layer was separated, dried over Na 2

SO

4 and concentrated to give a white solid residue. The residue was purified by column chromatography (0.5% WO 01/51499 PCT/AUO1/00028 - 58 MeOH/DCM, v/v) to give 2-acetamido-3,6-di-O-benzyl-2-deoxy 4-0-[4,6-0-benzylidene-2-0-pivaloyl-3-0-(2,3,4,6-tetra-0 (4-chlorobenzyl)-a-D-galactopyranosyl)-$-D galactopyranosyl)]- -D-glucopyranoside 58 (213mg, 76.3%). 5 Rf = 0.57 (66% ethylacetate/petroleum ethers, v/v), ES-MS m/z (ion, intensity) 1486.29 ([M+H]+ 100%) In a cognate experiment to experiment 58, compound 47 was substituted with compound 43 (the experiment employing resin 53 (425mg, 0.199mmol/g)), to afford 2-acetamido-3,6 10 di-0-benzyl-2-deoxy-4-0-[4,6-0-benzylidene-2-0-(4 chlorobenzoyl)-3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-cX-D galactopyranosyl)- -D-galactopyranosyl)]--D glucopyranoside 59 (96mg, 34%), Rf = 0.23 (1.64% methanol/dichloromethane, v/v), ES-MS m/z (ion, intensity) 15 1543.29 ([M+H]* 100%) In a further cognate experiment to experiment 58, compound 47 was substituted with compound 50 to afford 2 amino-3,6-di-0-benzyl-2-deoxy-4-0-[4,6-0-benzylidene-3-0 (2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D-galactopyranosyl)-$ 20 D-galactopyranosyl)]-$-D-glucopyranoside 60, Rf = 0.5 (1.96% methanol/dichloromethane, v/v), ES-MS m/z (ion, intensity) 1360.73 ([M+H]* 100%) Synthesis of 2-Acetamido-3,6-di-O-benzyl-2-deoxy-4-0-[4,6 25 0-benzyli-dene-3-O- (2,3,4, 6-tetra-O- (4-chl orobenzyl) -a-D galactopyranosyl) -p-D-gal actopyrano-syl) ] -#8-D glucopyranoside (61) 2-Acetamido-3,6-di-O-benzyl-2-deoxy-4-0-[4,6-0 benzyli-dene-2-0-pivaloyl-3-0-(2,3,4,6-tetra-O-(4 30 chlorobenzyl)-aX-D-galactopyranosyl)- -D-galactopyranosyl)] $-D-glucopyranoside 58 [288mg, 188pmol] was suspended in a solution of NaOMe/MeOH [0.13M, 10mL] to which was added acetonitrile [5mL]. The reaction was heated at 70 0 C until TLC indicated that the reaction had gone to completion (4-5 35 days). The reaction mixture was then concentrated and taken up in dichloromethane [20mL] and washed with 10% citric acid solution [2 x 20mL] and saturated brine solution [2 x WO 01/51499 PCT/AUO1/00028 - 59 20mL] . The organic layer was separated, dried over Na 2

SO

2 and the solvent removed in vacuo to provide a solid white residue. The residue was purified by preparative thin layer chromatography (eluent: 13% Acetone/DCM) to give 2 5 Acetamido-3, 6-di-O-benzyl-2-deoxy-4-0- [4, 6-O-benzyli-dene 3-0-(2,3,4,6-tetra-O-(4-chlorobenzyl)-a-D galactopyranosyl)-$-D-galactopyranosyl)]- -D glucopyranoside 61 [189mg, 69%]. Rf 0.24 (1.47% MeOH/DCM); ES-MS m/z (ion, intensity) 1403.29 ([M+H]*, 100%) 10 WO 01/51499 PCT/AUO1/00028 - 60 Synthesis and Immobilisation of Gal-a-(1-3)-Gal-3-(1-4) GlcNAc-Linker Conjugate. Scheme 9: Synthesis of Gal-a-(1-3)-Gal- -(1-4)-GlucNAc-conjugate 5 OH OH O OH OH HO O OH O O OH O 0_ HO 28 OH NHAc OH OH 30% NH 3

/H

2 0/NH 4

HCO

3 /45 0 C, 48 h OH O OH OH HO OA0o OH0 0 HO 62 OH NHAc NH2

OCN(CH

2

)

3 C1/MeOH/rt, 2 h OH OH I O OH OH H O H OHoo O-, HO 63 OH NHAc NHCONH(CH 2

)

3 c1 KSAc/Nal/H 2 0/80C, 2 h OH O OH O OH OH HO OH 64 OH NHAc NHCONH(Qm 2

)

3 SAc 1. NaOMe/MeOH/rt, I h/MeClAc/rt, 2h 2. NaOH/H 2 0/MeOH/rt, 6 bH* resin/30 min OH O OH i 0 OH OH k, Oo OH 65 OH O O 0 - HO OH -300 mg, pure by LC-MS NHA NHCONH(CH 2

)

3

SCH

2

CO

2

H

WO 01/51499 PCT/AUO1/00028 - 61 Example 11: Synthesis of Sugar-Linker Conjugate 2-Acetamido-2-deoxy-4 -0- [3-0- (c-D-galactopyranosyl) -$-D galactopyranosyl ]-D-glucopyranosyl amine (62) 5 A solution of 2-Acetamido-2-deoxy-4-0-[3-0-(a-D galactopyranosyl)-$-D-galactopyranosyl)-D-glucopyranose (1 g, 1.8 mmol) 28 and ammonium bicarbonate (0.15 g, 1.9 mmol) in 30% aqueous ammonia (20 mL) was left to stir at 400C for 48 h. The reaction mixture was then freeze dried to give 10 62 (1.0 g, -80% yield by tic) as a white solid. Tlc Rf 0.2 (AcN : water, 3 : 1) 1-N- (3-chloropropyl) -1-N' -urei do-2-acetamido-2-deoxy-4-0 15 [3-0-(a-D-galactopyranosy1)-#-D-galactopyranosyl]-D glucopyranoside (63) To a solution of 62 (0.35 g, 6.5 mmol) in methanol (5 mL), was added, 3-chloropropylisocyanate (0.1 g, 0.84 mmol). The reaction mixture was then left to stir at room 20 temperature overnight. The reaction contents was evaporated to dryness and the remaining residue was dissolved in water* (-3 mL) and loaded on to a C-18 Sep pack column (5 g). The column was eluted** with water (50 mL) followed by 25% methanol in water (50 mL). The 25 methanol fractions were combined and evaporated to dryness giving pure 63 (350 mg, -80% yield) as a white solid. Tlc Rf 0.6 (AcN : water, 3 : 1) M+H found 664 30 HPLC Rt 4.0 and 4.5 min for og3 anomers (linear gradient: 5% AcN to 20% AcN over 15 min, C-18 column) 1-N-(3-acetoxythiopropyl) -1-N' -ureido-2-acetamido-2-deoxy 35 4-0- [ 3-0- (a-D-galactopyranosyl) -/-D-galactopyranosyl] -D glucopyranoside (64) WO 01/51499 PCT/AUO1/00028 - 62 A mixture of 63 (0.2 g, 0.30 mmol), sodium iodide (0.1 g, 0.67 mmol) and potassium thioacetate (0.2 g, 1.74 mmol) in water (10 mL) was left to stir at 80 0 C for 2 h. The reaction mixture was then cooled to room temperature 5 and concentrated to 5 ml. The concentrate was loaded on to a C-18 Sep-pack column (5 g) which was then eluted with water (100 mL) followed by 25% methanol in water (100 mL). The methanol fractions were combined and evaporated to dryness to give pure 64 (0.18g, -85% yield) as a white 10 solid. Tlc Rf 0.6 (AcN : water, 3 : 1) M+H found 703 HPLC Rt 5.5 and 6.0 min for Wo3 anomers (linear gradient: 15 5% AcN to 20% AcN over 15 min, C-18 column) 1-N- [3- (methyl carboxymethythio) -propyl] -1-N' -urei do-2 ace tamido-2 -deoxy-4-O- [3-0- (a-D-galactopyranosyl) -#-D 20 galactopyranosyl]-D-glucopyranoside (65) To a solution of sodium methoxide (14 mg, 0.26 mmol) in methanol (3 mL), was added 64 (110 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 20 min and then methyl bromoacetate (50 mg, 0.30 mmol) was added. 25 The resultant mixture was left to stir at room temperature for 2 h. The reaction mixture was quenched with acetic acid (200 pL) and then evaporated to dryness. The residue was dissolved in water (2 mL) and loaded on to a C-18 Sep pack column (5 g). The column was eluted with water (50 3,0 ml) followed by 50% methanol in water (50 mL). The methanol fractions were combined and evaporated to dryness giving 65 (100.8 mg, 90% yield) as a white solid. Tlc Rf 0.65 (AcN : water, 3 : 1) 35 M+H found 734, M+Na found 755 WO 01/51499 PCT/AUO1/00028 - 63 1-N- [3- (carboxyme thylthio) -propyl] -1-N'-ureido-2acetami do 2-deoxy-4-O-[3-0-(a-D-galactopyranosyl)-/3-D galactopyranosyl] -D-glucopyranoside (66) A solution of 65 (300 mg, 0.41 mmol) and potassium 5 hydroxide (30 mg, 0.53 mmol) in 30% aqueous methanol (15 mL) was left to stir at room temperature for 4 h. The reaction mixture was diluted to 50 mL with methanol and then neutralised with IR-120 H" resin. The suspension was then filtered and the filtrate evaporated to dryness 10 leaving 66 (295 mg, 100% yield) as a white solid. Tlc Rf 0.30 (AcN : water, 3 : 1) M+H found 719 Notes 15 *Milli-Q-Water was used at all times **Flow rate was one drop/sec at all times WO 01/51499 PCT/AUO1/00028 - 64 Scheme 10: Coupling of Gal-ox-(1-3)-Gal- -(1-4)-GlucNAc-linker conjugate to propylamino-functionalised silica and hexylamino-functionalised Sepharose OH OH 0 OH OH OB OH 66 OH O O 0 HO OH NHAc NHCONH(CH2)SCH 2

CO

2

H

Si-O-(CH 2

)

3

-NH

2 (67) or Sepharose-(CH 2

)

6 -NI

H

2 0/EDC/NHS/rt, o'night OH OH O OH OH O O H OH O O H O 0_ HO OH NHAc NHCONH(CH 2

)

3

SCH

2

CONH(CH

2 )nOX 5 n = 3 for silica and 6 for Sepharose 10 x = Sepharose(69) or silica (68) Example 12: Immobilisation of Gal-a-(1-3)-Gal-$8-(1-4) GlucNAc-Linker Conjugate 15 Preparation of 0.3 mmol propylamido-FmocAla-functionalised silica (67) To a mixture of FMOC-Ala (2.65 g, 8.5 mmol) and HBTU (3.23 g, 8.5 mmol) in dry DMF (20 mL), was added DIPEA (1.1 g, 8.5 mmol). The mixture was shaken for 2 min and then left to stand for 15 min. The mixture was then added to a suspension of propylamino functionalised silica* (17 g) in dry DMF (20mL). The resultant mixture 20 was shaken end over end for 18 h at room temperature. The mixture was filtered and the silica washed with DMF (3 x 100 mL) followed by methanol (3 x 100 mL). The resin was resuspended in a mixture of methanol (100 mL) and acetic anhydride (50 mL) and then shaken for 2 h (negative ninhydrin test after this time). The suspension was WO 01/51499 PCT/AUO1/00028 - 65 filtered and the silica was then washed with methanol (4 x 100 mL) and dried. The loading of FMOC-Ala was found to be 0.3 mmol per gram** of silica. *Silica was washed with DIPEA prior to coupling. 5 **FMOC-Ala loading was quantitated by cleaving (20% piperidine in DMF) a known quantity of FMOC-Ala capped silica and determining the concentration from the UV absorption of the cleavage product at 290 nm against a standard curve. 10 Coupling of 66 to propylamido-Ala-functionalised silica (68) FMOC-Ala modified silica from above was cleaved by the standard method (20% piperidine in DMF, rt, 20 min) to give the corresponding free amino (-0.3 mmol loading) 15 functionalised silica. This was then used for the trisaccharide couplings described below. Loading 1, -20 mg of F per gram of Ala-capped silica: To NHS (235 mg, 2.08 mmol), was added a solution of 20 66 (100 mg, 0.139 mmol) and EDC.HC1 (2.15 g, 11.2 mmol) in water (10 mL). The resulting solution was added to a suspension of Ala-capped silica (5 g) in water (-10 mL). The suspension was left to shake at room temperature for 3 h, at which time no trisaccharide was present in the 25 filtrate, by tlc. The suspension was then drained, washed with water (4 x 50 ml), dilute sodium bicarbonate solution (3 x 50 ml) and again with water (3 x 50ml). The silica was then resuspended in methanol/acetic anhydride (30 ml, 3:1) and left to shake for 1 h (negative ninhydrin test 30 after this time). The suspension was then drained and the .silica washed with methanol (4 .x 50 ml) to give the trisaccharide capped silica. Loading 2, -5.0 mg of 66 per gram of Ala-capped silica: 35 66 (25 mg, 0.034 mmol), NHS (100 mg, 0.884 mmol), EDC.HC1 (1.2 g, 6.25 mmol), WO 01/51499 PCT/AUO1/00028 - 66 and Ala-capped silica (5 g). Prepared as described for loading 1. Loading 3, -0.5 mg of 66 per gram of Ala-capped silica: 5 66 (2.5 mg, 0.0034 mmol), NHS (30 mg, 0.265 mmol), EDC.HC1 (130 mg, 0.677 mmol), and Ala-capped silica (5 g). Prepared as described for loading 1. 10 Coupling of 66 to hexylamino-functionalised Sepharose (EAH Sepharose 4B) (69) Loading, -3.5 to 6.0 mg of 66 per mL of EAH Sepharose: 15 EAH Sepharose (5 mL) was washed with water (3 x 50 ml) and then suspended in water (5 ml). To the suspension a solution of 66 (94 mg, 0.131 mmol), EDC.HC1 (1.55 g, 8.10 mmol) and NHS (290 mg, 2.57 mmol) in water (15 mL) was 20 added. The reaction mixture was left to shake overnight at room temperature. Tlc of the filtrate showed no 66 present after this time. The reaction contents were drained and the resin was washed with water (3 x 50 mL). The modified Sepharose was then stored as a concentrated suspension in 25 5% ethanol in water (5 mL). It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and 30 methods described herein may be made without departing.from the scope of the inventive concept disclosed in this specification. References cited herein are listed on the following pages, and are incorporated herein by this reference.

WO 01/51499 PCT/AUO1/00028 - 67 REFERENCES Aug6, C. and Veyrieres, A., J.C.S. Perkin I, 1979 1825-1832 5 Boriello, S.P., J. Med. Microb., 1990 33 207-215 Burakoff, R., Zhao, L., Celifarco, A.J. et al, 10 Gastroenterology, 1995 109 348-354 Castex, F., Jouvert, S., Bastide, M. and Corthier, G. J. Med. Microbiol., 1994 40 102-109 15 Chacon-Fuertes, M.E. and Martin-Lomas, M. Carbohydrate Res., 1975 43 51-56 Eglow, R. et al. J. Clin. Invest., 1992 90 822-829 20 Garegg, P.J. and Oscarson, S. Carbohydrate Research, 1985 136 207-213 Good, H., Cooper, D.K.C. et al. 25 Transplant. Proc., 1992 24 559 Ichiro, Matsuo., Hiroshi, Fujimoto., Megumi, Isomura. and Katsumi, Ajisaka., Biorganic & Medicinal Chemistry Letters, 1997 7 (3) 255-258 30 Krivan, H.C., Clark, G.F., Smith, D.F. and Wilkins, T.D. Infect. Immun., 1986 53 573-581 Lemieux, R.U. and Driguez, H., 35 Journal of the American Chemical Society, 1975 97(14) 469 475 WO 01/51499 PCT/AUO1/00028 - 68 Matsuo, Ichiro; Fujimoto, Hiroshi; Isomura, Megumi and Ajisaki, Katsumi Bioorganic & Medicinal Chemistry Letters, 1997 7(3) 255-258 5 Milat, M-L., Zollo, P.A. and Sina?, P. Carbohydrate Research, 1982 100 263-271 Nilsson, K.G.I. Tetrahedron Letters, 1997 38 (1) 133-136 10 Schaubach, R., Hemberger, J. and Kinzy, W. Liebigs Ann. Chem., 1991 607-614 Simon, P.M., 15 DDT 1 (12) Dec 1996 Sinay, P. and Jacquinet, J.C. Tetrahedron, 1979 35 365-371 20 Smith, J.A. et al. J. Med. Microb., 1997 46 953-958 Sujino, Keiko., Malet, Charles., Hindsgaul, Ole. and Palcic, Monica M. 25 Carbohydrate Research, 1998 305 483-489 Takeo, Ken'ichi and Maeda, Hideaki J. Carbohydrate Chemistry, 1988 7(2) 309-316 30 Tong Zhu and Geert-Jan Boons J. Chem. Soc., Perkin Trans.I, 1998 857-861 Torres, J., Jennische, E., Lange, S. and Lonnroth, I., Gut, 1990 31 781-785 35 WO 01/51499 PCT/AUO1/00028 - 69 Vic, G., Chuong Hao Tran, Scigelova, M. and Crout, D.H.G. Chem. Commun., 1997 169-170

Claims (27)

1. 2. A protected monosaccharide building block of general 15 formula II: O O SMe 0 *."'ORl 2 II R OR in which R 3 is methoxy or methyl; R 1 is H, benzoyl, pivaloyl, , 4-chlorobenzoyl, acetyl, 20 chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4 acetamidobenzyl, or 4-azidobenzyl; and R 2 is H, Fmoc, benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 25 3, 4-methylenedioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl,
2. 4-acetamidobenzyl, or 4-azidobenzyl. 3. A protected monosaccharide building block according to claim 2, in which WO 01/51499 PCT/AU01/00028 - 71 R 3 is H, R1 is benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, benzyl, 3,4-methylene dioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4 acetamidobenzyl, or 4-azidobenzyl, and 5 R2 is Fmoc, benzoyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4 acetamidobenzyl, or 4-azidobenzyl, with the provisos that 10 (a) when R1 is acetyl, R 2 is not chloroacetyl or acetyl, and vice versa; (b) when R 2 is levulinoyl, R' is not benzoyl, and vice versa; and (c) when R' is benzoyl, R 2 is not benzoyl, and vice versa. 15 4. A protected monosaccharide building block according to claim 2 or claim 3, in which R 2 is Fmoc, and R' is benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, benzyl, 3,4 methylene-dioxybenzyl, 4-methoxybenzyl, 4-chlorobenzyl, 4 20 acetamidobenzyl, or 4-azidobenzyl.
3. 5. A protected monosaccharide building block according to any one of claims 2 to 4, in which the compound is of general formula III: 25 O 0 SMe , ,. I III O. " 0R' OR 2 in which R1 is pivaloyl, benzoyl, 4-chlorobenzoyl, 4 methoxybenzyl, or 3,4-methylenedioxybenzyl, and R2 is H, Fmoc, 4-chlorobenzoyl, acetyl, chloroacetyl, 30 levulinoyl, 4-methoxybenzyl, or 3,4-methylenedioxybenzyl, with the proviso that if R' is benzoyl, R 2 is not levulinoyl. WO 01/51499 PCT/AU01/00028 - 72 6. A protected monosaccharide building block according to claim 5, in which the compound is a galactopyranoside, R' is 4-chlorobenzoyl, pivaloyl or acetyl, and R2 is Fmoc or H. 5 7. A protected monosaccharide building block according to claim 5, in which R 1 is 4-chlorobenzoyl and R 2 is chloroacetyl.
4. 8. A protected monosaccharide building block according to claim 5, in which both R' and R 2 are 3,4 10 methylenedioxybenzyl.
5. 9. A galactopyranoside compound of general formula IV: R 1 0 0 SMe R 1 0 OR IV OR in which each R1 is independently 4-chlorobenzyl, 4 15 azidobenzyl, 4-N-acetamidobenzyl, 4-methylbenzyl, 3,4 methylenedimethoxybenzyl, or 2-nitrobenzyl.
6. 10. A galactopyranoside according to claim 9, in which each R 1 is 4-chlorobenzyl.
7. 11. A polyethyleneglycol (PEG)-linked monosaccharide of 20 general formula V: 0 0 O R 0n OR 3 V in which n is an integer from 1-5; 25 R' is a linking group or a group suitable for the formation of a covalent linkage; WO 01/51499 PCT/AUO1/00028 - 73 R2 is acetyl, 4-chlorobenzoyl, levulinoyl, pivaloyl, chloroacetate, benzoyl, 4-methybenzoyl; R 3 is H, Fmoc, benzoyl, pivaloyl, 4-chlorobenzoyl, acetyl, chloroacetyl, levulinoyl, 4-methylbenzoyl, 3,4 5 methylenedioxybenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl; and R 4 is methoxy, H, or methyl.
8. 12. A polyethyleneglycol (PEG)-linked monosaccharide according to claim 11, in which R 1 is selected from the 10 group consisting of halogen, azido, carboxylic acid, thiol, hydroxyl, thioester, xanthate, amido, and dithiocarbamate.
9. 13. A PEG-linked monosaccharide according to claim 11 or claim 12, in which n is 2, R' is thiobenzoate or thiobiphenylcarbonyl, R 2 is 4-chlorobenzoyl, R 3 is H, and R 4 15 is H.
10. 14. A compound of general formula VI: R 4 0 0 OR R O 4 O O'R'R 0 0 0 ~OR 3 7" 0 "'OR 5 7OR 6 V R I in which R 7 is H, methoxy or methyl; 20 R 1 is aryl, substituted aryl, benzyl, substituted benzyl, alkyl, substituted alkyl, PEG, or substituted PEG; R2 is acetamido or amino; R3 and R 4 are independently benzyl, substituted benzyl, silylether or acyl; 25 R 5 is 4-chlorobenzoyl, benzoyl, pivaloyl, acetyl, levulinoyl or 4-methylbenzoyl; and R6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3, 4-methylenedioxybenzyl, 4-methoxybenzyl, 4 30 acetamidobenzyl, or 4-azidobenzyl. WO 01/51499 PCT/AUO1/00028 - 74 15. A compound according to claim 14, in which the anomeric configuration of the the glucosamine moiety is a; R3 is benzyl, R4 is benzoyl and R7 is H, R2 is optionally acetamido, amino, or N-phthalimido, R 5 is optionally 4 5 chlorobenzoyl, benzoyl, pivaloyl, acetyl, levulinoyl or 4 methylbenzoyl, and R 6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3,4-methylenedioxybenzyl, 4 methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl. 10 16. A compound according to claim 14, in which the anomeric configuration of the the glucosamine moiety is P; R is benzyl and R7 is H, R2 is acetamido, amino, or N phthalimido; R 3 and R 4 are independently benzyl, substituted benzyl, silylether or acyl; R 5 is 4 15 chlorobenzoyl, benzoyl, pivaloyl, acetyl, levulinoyl or 4 methylbenzoyl, and R 6 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3,4-methylenedioxybenzyl, 4 methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl. 20 17. A compound according to claim 14, in which the anomeric configuration of the the glucosamine moiety is a; RI, R 3 , and R 4 are benzyl or substituted benzyl, and R7 is H, R2 is acetamido, amino, or N-phthalimido, R is pivaloyl, 4-chlorobenzoyl, benzoyl, or levulinoyl, and R 6 25 is a substituted or unsubstituted pyranosyl or furanosyl sugar, H, Fmoc, acetyl, chloroacetyl, levulinoyl, 3,4 methylenedioxybenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, or 4-azidobenzyl, with the proviso that when R 3 and R 4 are benzyl, R 5 is not acetyl or benzoyl. 30 18. A compound according to claim 14, in which the anomeric configuration of the the glucosamine moiety is 3; R is benzyl, R 2 is amino or acetamido, R 3 and R 4 are benzyl, R 5 is 4-chlorobenzoyl, pivaloyl or acetyl, R 6 is Fmoc or H, and R 7 is H. 35 19. A compound according to claim 14, in which the anomeric configuration of the the glucosamine moiety is a; RK is benzyl, R 2 is acetamido, R 3 is benzyl, R 4 is benzoyl WO 01/51499 PCT/AUO1/00028 - 75 or benzyl, R 5 is 4-chlorobenzoyl, R 6 is H or 4-chloroacetyl and R 7 is H.
11. 20. A compound according to claim 14, in which the compound is a trisaccharide of General Formula VII: 5 Ph 0 OR' CIBnO O0 0 OBn 0 1 CIBnO .'0 O 'NHR 2 - OR OBn CIBnO OBnCI VII in -which R is H or acetyl; R1 is hydrogen, benzyl, benzoyl or p-chlorobenzoyl; and R2 is hydrogen, 4-chloro-benzoyl, 10 acetyl, benzoyl or pivaloyl.
12. 21. A compound according to claim 20, in which the anomeric configuration of the reducing end of the trisaccharide is a, R is acetyl, R' is benzoyl, 4 chlorobenzoyl or H, and R 2 is 4-chlorobenzoyl or H. 15 22. A compound according to claim 20, in which the anomeric configuration of the reducing end of the trisaccharide is P, R is acetyl or H, R' is benzyl, and R 2 is H, 4-chlorobenzoyl, pivaloy.1 or acetyl.
13. 23. A compound of general .formula VIII: R 4 0 R 7 0 R 3 0 0 R 6 0 "00 OR 5 OR 5 OR 2 X-R' VIII 20 in which R 5 , R 6 and R7 are independently H, 4 chlorobenzyl, 4-methoxybenzyl, 4-methylbenzyl, 4 acetamidobenzyl, azidobenzyl or 3, 4-methylenedioxybenzyl; WO 01/51499 PCT/AUO1/00028 - 76 X is 0, S, or N; R 1 is alkyl, substituted alkyl, aryl, substituted aryl, PEG or substituted PEG; R 2 is levulinoyl, 4-chlorobenzoyl, benzoyl, 4 5 methylbenzoyl, acetyl or pivaloyl; and R 3 and R 4 either combine to form a benzylidene ring, which may optionally be substituted at the 4 position by mthyl or methoxy, or R 3 and R 4 are independently H, benzyl or substituted benzyl. 10 24. A compound according to claim 23, in which R 5 is 4 chlorobenzyl, 4-methoxybenzyl, 4-methylbenzyl, 4 acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl, and R 6 and R 7 combine to form a benzylidene or substituted benzylidene ring; X is 0, S, or N; R1 is alkyl, substituted 15 alkyl, aryl, substituted aryl, PEG, substituted PEG, acyl or substituted acyl; and R 2 is levulinoyl, 4 chlorobenzoyl, benzoyl, 4-methylbenzoyl, acetyl or pivaloyl.
14. 25. A compound according to claim 23, in which X is 20 oxygen; R 1 is 3,4-methylenedioxybenzyl; R 2 is H, 4 chlorobenzoyl, pivaloyl, acetyl, levulinoyl, benzoyl or chloroacetyl; R 3 and R 4 either combine to become a benzylidene ring or are independently H, benzyl or substituted benzyl; and R , R 6 and R 7 may be H, benzyl, 4 25 chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl.
15. 26. A compound according to claim 23, in which X is oxygen; R3 is 2-[2-(2-thiobenzoyl)-ethoxy)ethyl or 2-[2-(2 thiobiphenylcabonyl)ethoxy); R 2 is H, 4-chlorobenzoyl, 30 pivaloyl, acetyl, levulinoyl, benzoyl or chloroacetyl; R 3 and R 4 combine to form a benzylidene ring, or are independently H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl; R 5 is H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4 35 acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl; and R 6 and R 7 combine to become a benzylidene ring or are WO 01/51499 PCT/AUO1/00028 - 77 independently H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3,4-methylenedioxybenzyl.
16. 27. A compound according to claim 23, in which X is sulphur; R is alkyl, substituted alkyl, aryl or 5 substituted aryl; R 3 and R 4 combine to form a benzylidene ring; R 5 , R 6 and R 7 are benzyl; and R 2 is levulinoyl, 4 chlorobenzoyl, benzoyl, acetyl or pivaloyl, with the proviso that when R' is phenyl, R 2 is not levulinoyl. 10 28. A compound according to claim 23, in which X is oxygen; R' is 2-[2-(2-thiobenzoyl)ethoxy)ethyl or 2-[2-(2 thiobiphenylcabonyl)ethoxy); R2 is H or 4-chlorobenzoyl; R 3 and R 4 are H or combine to form a benzylidene ring; R 5 is H or 3,4-methylenedioxybenzyl; and R 6 and R 7 are both H, or 15 combine to form a benzylidene ring.
17. 29. A compound according to claim 23, in which X is S, R' is methyl; R 2 is 4-chlorobenzoyl; R 3 and R 4 combine to form a benzylidene ring; and R 5 , R 6 and R 7 are each 4 chlorobenzyl. 20 30. A compound according to claim 23, in which X is oxygen; R is 3,4-methylenedioxybenzyl; R 2 is 4 chlorobenzoyl or H; R 3 and R 4 combine to form a benzylidene ring or are both H; and R5, R and R 7 are independently 4 chlorobenzyl or H. 25 31. A compound of general formula IX: 0 R 4 0 0 O R 3 0 IX OR 2 in which R' is 4-chlorobenzoyl, pivaloyl, acetyl, levulinoyl, benzoyl or chloroacetyl; WO 01/51499 PCT/AUO1/00028 - 78 2' R is H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4 acetamidobenzyl, azidobenzyl, 3, 4-methylenedioxybenzyl, Fmoc, levulinoyl, acetyl or chloroacetyl; and R3 and R 4 combine to form a benzylidene ring, or are 5 independently H, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 4-acetamidobenzyl, azidobenzyl or 3, 4-methylenedioxybenzyl.
18. 32. A -compound according to claim 31, in which R 1 is 4 chlorobenzoyl, R 2 is H, and R 3 and R 4 combine to form a benzylidene ring. 10 33. A polyethyleneglycol(PEG)-linked disaccharide of General Formula X or a trisaccharide of General Formula XI: OH OH 0OH OH HO OH O R 0 &' 0 _0 0 8'R OH HO 0 AcHN OH OH 0 0 O OH OH n HO o OH '0 0 ~ OH O 15 XI in which R is hydrogen or acyl, and n is ai eager 20 of from 1 to 3.
19. 34. A compound of Formula XI according to claim 33, which is 2- [2- (2-thiobiphenylcarbor X :hoxy] -ethyl 3-0- (oX-D galactopyranosyl)-ca-galactopyranoside. WO 01/51499 PCT/AUO1/00028 - 79 35. A compound of general formula XII: 0 HO O NH NH,'(CH2)3''S'CH2 XNH-(CH2)-O '" NHAc OH ,0"H HO OH HQ.- OH OH OH OH 0 XII OH 5 in which X is a solid support, and n is an integer of from 3 to 6.
20. 36. A compound according to claim 35, in which X is Sepharose.
21. 37. A compound according to claim 35, in which X is 10 silica gel.
22. 38. A method of synthesis of a disaccharide or trisaccharide, comprising .the step of using a compound according to any one of claims 1 to 32 as an intermediate.
23. 39. A method according to claim 38, in which the 15 disaccharide or trisaccharide is selected from the group consisting of (a) a compound of General Formula X, General Formula XI or General Formula XII; (b) a-D-galactopyranosyl- (1-+3) -J-D-galactopyranosyl 20 (1-+4) -N-acetyl-D-glucosamine (Gala (1-+3) Galp (1--4) GlcNAc); (c) x-D-galactopyranosyl- (1-+3) -f-D-galactopyranose (Gala (1-+3) Gal); and (d) P-D-galactopyranosyl- (1-+4) -N-acetyl-D-glucosamine 25 (Galp (1-4) GlcNAc) .
24. 40. A method according to claim 38 or claim 39, in which the compound is of General Formula X or XI, and the intermediate compound is of General Formula V. WO 01/51499 PCT/AUO1/00028 - 80 41. A method according to claim 38, in which the compound is of General Formula VI, and the intermediate compound is of General Formula I.
25. 42. A method of preventing or reducing a hyperacute 5 rejection response associated with xenotransplantation, comprising the step of administering an effective dose of thioalkyl Gala-(1-+3)Gal or thioalkyl Gala (1-+3)Gal (1--+4)GlcNAc to a subject in need of such treatment. 10 43. A method of preventing or reducing hyperacute rejection associated with xenotransplantation, comprising the steps of a) removing plasma from a patient who is to undergo xenotransplantation; 15 b) exposing the plasma to thioalkyl Gala(1-+3)Gal or thioalkyl Galc(l->3)Gal$(l-+4)GlcNAc linked to a solid support, and c) reinfusing the thus-treated plasma into the patient. 20 44. A method of depleting anti-Gala(1-+3)Gal antibodies from a plasma or serum sample, comprising the step of exposing the plasma or serum to thioalkyl Galcx(l-+3)Gal or thioalkyl Gala(1-+3)GalP(1-+4)GlcNAc linked to a solid support. 25 45. A method of treatment of C. difficile infection, comprising the step of administering an effective amount of cx-D-galactopyranosyl- (1-43) -- D-galacto-pyranosyl- (1->4) -N acetyl-D-glucosamine (Gala(1-+3)Gal (1-+4)GlcNAc) or of thioalkyl GalX(1-+3)GalS(1-+4)GlcNAc to a subject in need 30 of such treatment.
26. 46. A method according to claim 45, in which the GalX(1-+3)Gal3 (1-+4)GlcNAc) or thioalkyl Gala(1-43)Gal$(1-+-4)GlcNAc, is linked to a solid support.
27. 47.A method according to claim 45, in which the solid 35 support is a multidentate ligand or a dendrimer compound.